LOCUS pKLAC-malE 10153 bp DNA circular 18-OCT-2007 DEFINITION Control vector pKLAC-malE. ACCESSION VERSION KEYWORDS . SOURCE Control vector pKLAC-malE ORGANISM Control vector pKLAC-malE other sequences; artificial sequences; vectors. REFERENCE 1 (bases 1 to 10153) AUTHORS Colussi,P.A. and Taron,C.H. TITLE Kluyveromyces lactis LAC4 promoter variants that lack function in bacteria but retain full function in K. lactis JOURNAL Appl. Environ. Microbiol. 71 (11), 7092-7098 (2005) PUBMED 16269745 REFERENCE 2 (bases 1 to 10153) AUTHORS Colussi,P.A. and Taron,C.H. TITLE Direct Submission JOURNAL Submitted (18-OCT-2007) Research Department, New England Biolabs, 240 County Road, Ipswich, MA 01938, USA FEATURES Location/Qualifiers source 1..10153 /organism="Control vector pKLAC-malE" /mol_type="other DNA" gene 14..1384 /gene="alpha-MF/malE fusion" CDS 14..1384 /gene="alpha-MF/malE fusion" /codon_start=1 /product="Kluyveromyces lactis alpha mating factor pre-pro secretion leader fused to Escherichia coli maltose binding protein (MBP)" /translation="MKFSTILAASTALISVVMAAPVSTETDIDDLPISVPEEALIGFI DLTGDEVSLLPVNNGTHTGILFLNTTIAEAAFADKDDLEKRMKTEEGKLVIWINGDKG YNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSG LLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEI PALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKA GLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLP TFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKS YEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDA QTNSSS" sig_peptide 14..268 /gene="alpha-MF/malE fusion" /note="directs fusion protein to the general secretory pathway; cleaved off in the Golgi apparatus by the Kex protease." terminator 1417..1999 /note="Kluyveromyces lactis LAC4 transcription terminator; stops transcription from the alpha-MF/target gene fusion" promoter 2056..2758 /note="Saccharomyces cerevisiae ADH1 promoter; drives amdS transcription" gene 2759..4405 /gene="amdS" CDS 2759..4405 /gene="amdS" /note="selectable marker allowing growth on acetamide as a sole nitrogen source" /codon_start=1 /product="acetamidase" /translation="MPQSWEELAADKRARLAKTIPDEWKVQTLPAEDSVIDFPKKSGI LSEAELKITEASAADLVSKLAAGELTSVEVTLAFCKRAAIAQQLTNCAHEFFPDAALA QARELDEYYAKHKRPVGPLHGLPISLKDQLRVKGYETSMGYISWLNKYDEGDSVLTTM LRKAGAVFYVKTSVPQTLMVCETVNNIIGRTVNPRNKNWSCGGSSGGEGAIVGIRGGV IGVGTDIGGSIRVPAAFNFLYGLRPSHGRLPYAKMANSMEGQETVHSVVGPITHSVED LRLFTKSVLGQEPWKYDSKVIPMPWRQSESDIIASKIKNGGLNIGYYNFDGNVLPHPP ILRGVETTVAALAKAGHTVTPWTPYKHDFGHDLISHIYAADGSADVMRDISASGEPAI PNIKDLLNPNIKAVNMNELWDTHLQKWNYQMEYLEKWREAEEKAGKELDAIIAPITPT AAVRHDQFRYYGYASVINLLDFTSVVVPVTFADKNIDKKNESFKAVSELDALVQEEYD PEAYHGAPVAVQVIGRRLSEERTLAIAEEVGKLLGNVVTP" promoter 5114..5694 /note="modified Kluyveromyces lactis LAC4 promoter 5' end" misc_feature 5365..5376 /note="BstXI restriction site (linearization, alternate)" misc_feature 5694..5699 /note="SacII restriction site (linearization)" rep_origin complement(6148..6736) /note="pMB1 origin of replication (counterclockwise) (RNAII -35 to RNA/DNA switch point)" gene complement(6907..7767) /gene="bla" CDS complement(6907..7767) /gene="bla" /note="ampR (confers resistance to ampicillin)" /codon_start=1 /product="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" promoter 8521..10153 /note="modified Kluyveromyces lactis LAC4 promoter 3' end" misc_feature 8521..8526 /note="SacII restriction site (linearization)" misc_feature 8578..8589 /note="BstXI restriction site (linearization, alternate)" BASE COUNT 2714 a 2390 c 2419 g 2630 t ORIGIN 1 aagcttgaaa aaaatgaaat tctctactat attagccgca tctactgctt taatttccgt 61 tgttatggct gctccagttt ctaccgaaac tgacatcgac gatcttccaa tatcggttcc 121 agaagaagcc ttgattggat tcattgactt aaccggggat gaagtttcct tgttgcctgt 181 taataacgga acccacactg gtattctatt cttaaacacc accatcgctg aagctgcttt 241 cgctgacaag gatgatctcg agaaaagaat gaaaactgaa gaaggtaaac tggtaatctg 301 gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga 361 taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt 421 tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta 481 cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta 541 tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt 601 tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga 661 agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa 721 cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa 781 gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc 841 gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta 901 ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg 961 ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt 1021 caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag 1081 tccgaacaaa gagctggcga aagagttcct cgaaaactat ctgctgactg atgaaggtct 1141 ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga 1201 gttggcgaaa gatccacgta ttgccgccac catggaaaac gcccagaaag gtgaaatcat 1261 gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc 1321 cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta attcgagctc 1381 gtaagcggcc gcttaattaa ggccttgaat cgagaattta tacttagata agtatgtact 1441 tacaggtata tttctatgag atactgatgt atacatgcat gataatattt aaacggttat 1501 tagtgccgat tgtcttgtgc gataatgacg ttcctatcaa agcaatacac ttaccaccta 1561 ttacatgggc caagaaaata ttttcgaact tgtttagaat attagcacag agtatatgat 1621 gatatccgtt agattatgca tgattcattc ctacaacttt ttcgtagcat aaggattaat 1681 tacttggatg ccaataaaaa aaaaaaacat cgagaaaatt tcagcatgct cagaaacaat 1741 tgcagtgtat caaagtaaaa aaaagatttt cactacatgt tccttttgaa gaaagaaaat 1801 catggaacat tagatttaca aaaatttaac caccgctgat taacgattag accgttaagc 1861 gcacaacagg ttattagtac agagaaagca ttctgtggtg ttgccccgga ctttcttttg 1921 cgacataggt aaatcgaata ccatcatact atcttttcca atgactccct aaagaaagac 1981 tcttcttcga tgttgtatac gttggagcat agggcaagaa ttgtggcttg agatcatcct 2041 tttgttgttt ccgggtgtac aatatggact tcctcttttc tggcaaccaa acccatacat 2101 cgggattcct ataatacctt cgttggtctc cctaacatgt aggtggcgga ggggagatat 2161 acaatagaac agataccaga caagacataa tgggctaaac aagactacac caattacact 2221 gcctcattga tggtggtaca taacgaacta atactgtagc cctagacttg atagccatca 2281 tcatatcgaa gtttcactac cctttttcca tttgccatct attgaagtaa taataggcgc 2341 atgcaacttc ttttcttttt ttttcttttc tctctccccc gttgttgtct caccatatcc 2401 gcaatgacaa aaaaatgatg gaagacacta aaggaaaaaa ttaacgacaa agacagcacc 2461 aacagatgtc gttgttccag agctgatgag gggtatctcg aagcacacga aactttttcc 2521 ttccttcatt cacgcacact actctctaat gagcaacggt atacggcctt ccttccagtt 2581 acttgaattt gaaataaaaa aaagtttgct gtcttgctat caagtataaa tagacctgca 2641 attattaatc ttttgtttcc tcgtcattgt tctcgttccc tttcttcctt gtttcttttt 2701 ctgcacaata tttcaagcta taccaagcat acaatcaagg aattccggat ccgccaccat 2761 gcctcaatcc tgggaagaac tggccgctga taagcgcgcc cgcctcgcaa aaaccatccc 2821 tgatgaatgg aaagtccaga cgctgcctgc ggaagacagc gttattgatt tcccaaagaa 2881 atcggggatc ctttcagagg ccgaactgaa gatcacagag gcttccgctg cggatcttgt 2941 gtccaagctg gcggccggag agttgacctc ggtggaagtt acgctagcat tctgtaaacg 3001 ggcagcaatc gcccagcagt taacaaactg cgcccacgag ttcttccctg acgccgctct 3061 cgcgcaggca agggaactcg atgaatacta cgcaaagcac aagagacccg ttggtccact 3121 ccatggcctc cccatctctc tcaaagacca gcttcgagtc aagggctacg aaacatcaat 3181 gggctacatc tcatggctaa acaagtacga cgaaggggac tcggttctga caaccatgct 3241 ccgcaaagcc ggtgccgtct tctacgtcaa gacctctgtc ccgcagaccc tgatggtctg 3301 cgagacagtc aacaacatca tcgggcgcac cgtcaaccca cgcaacaaga actggtcgtg 3361 cggcggcagt tctggtggtg agggtgcgat cgttgggatt cgtggtggcg tcatcggtgt 3421 aggaacggat atcggtggct cgattcgagt gccggccgcg ttcaacttcc tgtacggtct 3481 aaggccgagt catgggcggc tgccgtatgc aaagatggcg aacagcatgg agggtcagga 3541 gacggtgcac agcgttgtcg ggccgattac gcactctgtt gaggacctcc gcctcttcac 3601 caaatccgtc ctcggtcagg agccatggaa atacgactcc aaggtcatcc ccatgccctg 3661 gcgccagtcc gagtcggaca ttattgcctc caagatcaag aacggcgggc tcaatatcgg 3721 ctactacaac ttcgacggca atgtccttcc acaccctcct atcctgcgcg gcgtggaaac 3781 taccgtcgcc gcactcgcca aagccggtca caccgtgacc ccgtggacgc catacaagca 3841 cgatttcggc cacgatctca tctcccatat ctacgcggct gacggcagcg ccgacgtaat 3901 gcgcgatatc agtgcatccg gcgagccggc gattccaaat atcaaagacc tactgaaccc 3961 gaacatcaaa gctgttaaca tgaacgagct ctgggacacg catctccaga agtggaatta 4021 ccagatggag taccttgaga aatggcggga ggctgaagaa aaggccggga aggaactgga 4081 cgccatcatc gcgccgatta cgcctaccgc tgcggtacgg catgaccagt tccggtacta 4141 tgggtatgcc tctgtgatca acctgctgga tttcacgagc gtggttgttc cggttacctt 4201 tgcggataag aacatcgata agaagaatga gagtttcaag gcggttagtg agcttgatgc 4261 cctcgtgcag gaagagtatg atccggaggc gtaccatggg gcaccggttg cagtgcaggt 4321 tatcggacgg agactcagtg aagagaggac gttggcgatt gcagaggaag tggggaagtt 4381 gctgggaaat gtggtgactc catagcccgg ggggggctcg atcccctcgc gagttggttc 4441 agctgctgcc tgaggctgga cgacctcgcg gagttctacc ggcagtgcaa atccgtcggc 4501 atccaggaaa ccagcagcgg ctatccgcgc atccatgccc ccgaactgca ggagtgggga 4561 ggcacgatgg ccgctttggt cgatctagat tacgtggaag aaaggtagta aaagtagtag 4621 tataagtagt aaaaagaggt aaaaagagaa aaccggctac atactagaga agcacgtaca 4681 caaaaactca taggcacttc atcatacgac agtttcttga tgcattataa tagtgtatta 4741 gatattttca gaaatatgca tagaacctcc tcttgccttt actttttata catagaacat 4801 tggcagattt acttacacta ctttgtttct acgccatttc ttttgttttc aacacttaga 4861 caagttgttg agaaccggac tactaaaaag caatgttccc actgaaaatc atgtacctgc 4921 aggataataa ccccctaatt ctgcatcgat ccagtatgtt tttttttctc tactcatttt 4981 tacctgaaga tagagcttct aaaacaaaaa aaatcagtga ttacatgcat attgtgtgtt 5041 ctagtaacca aaggaaagga acagatagat aaaattccga gactgtcaaa ttaggttttt 5101 ttcttttttt ttggcgggag tcagtgggcc gaaatatgtt cttggcctag aacttaatct 5161 ggtttgatca tgccaatact tgcctgagtg cccgactttt tgcccaccct cttgccttct 5221 gtcatccttc aaaacccacc tgttttccag ccgtatcttc gctcgcatct acacatactg 5281 tgccatatct tgtgtgtagc cggacgtgac tatgaccaaa aacaaacaag gagaactgtt 5341 cgccgatttg taacactcct gcatccatcc aagtgggtat gcgctatgca atgttaagct 5401 aggtcaggtc agaccaggtc caaggacagc aacttgactg tatgcaacct ttaccatctt 5461 tgcacagaac atacttgtag ctagctagtt acacttatgg accgaaaagg caccccacca 5521 tgtctgtccg gctttagagt acggccgcag accgctgatt tgccttgcca agcagtagtc 5581 acaatgcatc gcatgagcac acgggcacgg gcacgggcac aggaaccatt ggcaaaaata 5641 ccagatacac tataccgacg tatatcaagc ccaagtttaa aattcctaaa tttccgcggc 5701 tacttttcaa ttccctatag tgagtcgtat taaattcgta atcatgtcat agctgtttcc 5761 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 5821 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 5881 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 5941 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 6001 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 6061 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 6121 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 6181 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 6241 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 6301 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 6361 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 6421 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 6481 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 6541 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 6601 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 6661 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 6721 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 6781 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 6841 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 6901 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 6961 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 7021 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 7081 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 7141 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 7201 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 7261 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 7321 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 7381 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 7441 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 7501 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 7561 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 7621 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 7681 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 7741 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 7801 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 7861 aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta gcggcgcatt 7921 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 7981 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 8041 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 8101 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 8161 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 8221 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc 8281 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt 8341 aacgcttaca atttccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg 8401 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt 8461 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgccaagctc 8521 ccgcggggat cgactcataa aatagtaacc ttctaatgcg tatctattga ctaccaacca 8581 ttagtgtggt tgcagaaggc ggaattctcc cttcttcgaa ttcagcttgc tttttcattt 8641 tttattttcc atttttcagt ttttgtttgt gtcgaattta gccagttgct tctccaagat 8701 gaaaaaaacc cctgcgcagt ttctgtgctg caagatccta atcgactttt ccacccccca 8761 caaaagtaaa tgttcttttg ttacattcgc gtgggtagct agctccccga atcttcaaag 8821 gacttaggga ctgcactaca tcagagtgtg ttcacctggt ttgctgcctg gtttgaaaga 8881 aaagagcagg gaactcgcgg gttcccggcg aataatcatg cgatagtcct ttggccttcc 8941 aagtcgcatg tagagtagac aacagacagg gagggcagga aggatctttc actgagatcc 9001 tgtatcttgt tgggtaagtc ggatgaaagg ggaatcgtat gagattggag aggatgcgga 9061 agaggtaacg ccttttgtta acttgtttaa ttattatggg gcaggcgaga gggggaggaa 9121 tgtatgtgtg tgaggcgggc gagacggagc catccaggcc aggtagaaat agagaaagcc 9181 gaatgttaga caatatggca gcgtagtaga gtaggtaggt aggcaagtac tgctagcaaa 9241 gaggagaagg gtaagctcac tcttcgcatt ccacaccgtt agtgtgtcag tttgaacaaa 9301 aaaacaatca tcataccaat tgatggactg tggactggct tttggaacgg cttttcggac 9361 tgcgattatt cgtgaggaat caaggtagga atttggtcat atttacggac aacagtgggt 9421 gattcccata tggagtagga aaacgagatc atggtatcct cagatatgtt gcggaattct 9481 gttcaccgca aagttcaggg tgctctggtg ggtttcggtt ggtctttgct ttgcttctcc 9541 cttgtcttgc atgttaataa tagcctagcc tgtgagccga aacttagggt aggcttagtg 9601 ttggaacgta catatgtatc acgttgactt ggtttaacca ggcgacctgg tagccagcca 9661 tacccacaca cgttttttgt atcttcagta tagttgtgaa aagtgtagcg gaaatttgtg 9721 gtccgagcaa cagcgtcttt ttctagtagt gcggtcggtt acttggttga cattggtatt 9781 tggactttgt tgctacacca ttcactactt gaagtcgagt gtgaagggta tgatttctag 9841 tggtgaacac ctttagttac gtaatgtttt cattgctgtt ttacttgaga tttcgattga 9901 gaaaaaggta tttaatagct cgaatcaatg tgagaacaga gagaagatgt tcttccctaa 9961 ctcgaaaggt atatgaggct tgtgtttctt aggagaatta ttattctttt gttatgttgc 10021 gcttgtagtt ggaaaaggtg aagagacaaa agctggaatt gtgagcggat aacaagctca 10081 acacttgaaa tttaggaaag agcagaattt ggcaaaaaaa ataaaaaaaa aataaacaca 10141 catactcatc gag //