LOCUS Exported 8007 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION vector for knocking out ald1 in Komagataella phaffii. ACCESSION . VERSION . KEYWORDS pUC_delta ald1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8007) TITLE Engineered ethanol-driven biosynthetic system for improving production of acetyl-CoA derived drugs in Crabtree-negative yeast REFERENCE 2 (bases 1 to 8007) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8007 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 26..45 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 239..261 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 253..270 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 254..270 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 1704..4238 /codon_start=1 /gene="Pichia pastoris HIS4" /product="multifunctional enzyme, required for histidine biosynthesis" /label=PpHIS4 /note="auxotrophic marker for Pichia pastoris" /translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVETCP GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI" primer_bind complement(5661..5677) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5661..5677) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5674..5696) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5685..5701 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(5709..5739) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5754..5775 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5892..5909) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6063..6651) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6143..6162) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6822..7682) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7445..7464 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7683..7787) /gene="bla" /label=AmpR promoter primer_bind 7855..7873 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(7911..7933) /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 61 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg 121 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc 181 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc 241 cagtcacgac gttgtaaaac gacggccagt gccaagcttg catgcctgca gaaatcaatc 301 gtcagttcaa tcaagacaac ccctcaggtg gtgttaatgc accgatatta atagttgacc 361 cacggcatga aacgtctaat gacgctgaga gaagaccaga tgctcctgcg actcaggctc 421 aacagtcttt ctcctcgtcc tcgacaaagt ttttgtctac tccctcctcg tctgttactc 481 ctcctttaga tactcatacc cgttcccaag caagttggaa gcaggcctgg ggagtttttc 541 aaagcattct ttccaaagct cagcccagcc gctctactgg agatgcaatt gccacaacta 601 attcggactc tcattcgcaa aagaagactg gttcgatgct ggtcaaacaa gatgcttcac 661 cacctcaacg cgataaaacc tttgtacatg gatatgaaca agatccccct tccagcggac 721 aattggagac cccaaaagca atcacaaaag taggactaac tagcgaccat tcaagtttat 781 caaggcgaac acccataatg gaatgttcca acaagagttc accttcagca aacggcaaca 841 gcactacaca aagtacacca gagagtaatt atacctcttt aatccggctc tcaagcatca 901 aagataaacc ttattttaca gagccacgat cctttcctgg aaaccatcta cggccagcac 961 agtcttcagt ggccaacgat ctcaacgaga gcacaggtga aacgttgcga aggacccatt 1021 cccatccttc agctgccgca cagaatgaat cattctcgct cgaccccagt cgtcaaaaga 1081 gaaaggaaaa cgacgacact tcttcacatc caaagagaag acgcacaatg actaggggtg 1141 acatagtaaa gagcgtgaga gcattacaca acccgttagg tcatgtagag agaatcatgg 1201 ctgcctacaa taagaacacg actaataata agatttttca ctcatgatga tcattgaacg 1261 ttaataatat ataaggtcat aaatactcag ttcagaattg gttaattggt tgtaacactg 1321 gcagagcatt acgctgactt gacgggacgg cggctttgtt gaataaatcg aacttttgct 1381 gagttgaagg atcagatcac gcatcttccc gacaacgcag accgttccgt ggcaaagcaa 1441 aagttcaaaa tcaccaactg gtccacctac aacaaagctc tcatcaaccg tggctccctc 1501 actttctggc tggatgatgg ggcgattcag gcctggtatg agtcagcaac accttcttca 1561 cgaggcagac ctcagcgccc ccccccccct gcaggtcggc cacgatgcgt ccggcgtaga 1621 ggatctcctg atgactgact cactgataat aaaaatacgg cttcagaatt tctcaagact 1681 acactcactg tccgacttca agtatgacat ttcccttgct acctgcatac gcaagtgttg 1741 cagagtttga taattccttg agtttggtag gaaaagccgt gtttccctat gctgctgacc 1801 agctgcacaa cctgatcaag ttcactcaat cgactgagct tcaagttaat gtgcaagttg 1861 agtcatccgt tacagaggac caatttgagg agctgatcga caacttgctc aagttgtaca 1921 ataatggtat caatgaagtg attttggacc tagatttggc agaaagagtt gtccaaagga 1981 tgatcccagg cgctagggtt atctatagga ccctggttga taaagttgca tccttgcccg 2041 ctaatgctag tatcgctgtg cctttttctt ctccactggg cgatttgaaa agtttcacta 2101 atggcggtag tagaactgtt tatgcttttt ctgagaccgc aaagttggta gatgtgactt 2161 ccactgttgc ttctggtata atccccatta ttgatgctcg gcaattgact actgaatacg 2221 aactttctga agatgtcaaa aagttccctg tcagtgaaat tttgttggcg tctttgacta 2281 ctgaccgccc cgatggtcta ttcactactt tggtggctga ctcttctaat tactcgttgg 2341 gcctggtgta ctcgtccaaa aagtctattc cggaggctat aaggacacaa actggagtct 2401 accaatctcg tcgtcacggt ttgtggtata aaggtgctac atctggagca actcaaaagt 2461 tgctgggtat cgaattggat tgtgatggag actgcttgaa atttgtggtt gaacaaacag 2521 gtgttggttt ctgtcacttg gaacgcactt cctgttttgg ccaatcaaag ggtcttagag 2581 ccatggaagc caccttgtgg gatcgtaaga gcaatgctcc agaaggttct tataccaaac 2641 ggttatttga cgacgaagtt ttgttgaacg ctaaaattag ggaggaagct gatgaacttg 2701 cagaagctaa atccaaggaa gatatagcct gggaatgtgc tgacttattt tattttgcat 2761 tagttagatg tgccaagtac ggtgtgacgt tggacgaggt ggagagaaac ctggatatga 2821 agtccctaaa ggtcactaga aggaaaggag atgccaagcc aggatacacc aaggaacaac 2881 ctaaagaaga atccaaacct aaagaagtcc cttctgaagg tcgtattgaa ttgtgcaaaa 2941 ttgacgtttc taaggcctcc tcacaagaaa ttgaagatgc ccttcgtcgt cctatccaga 3001 aaacggaaca gattatggaa ttagtcaaac caattgtcga caatgttcgt caaaatggtg 3061 acaaagccct tttagaacta actgccaagt ttgatggagt cgctttgaag acacctgtgt 3121 tagaagctcc tttcccagag gaacttatgc aattgccaga taacgttaag agagccattg 3181 atctctctat agataacgtc aggaaattcc atgaagctca actaacggag acgttgcaag 3241 ttgagacttg ccctggtgta gtctgctctc gttttgcaag acctattgag aaagttggcc 3301 tctatattcc tggtggaacc gcaattctgc cttccacttc cctgatgctg ggtgttcctg 3361 ccaaagttgc tggttgcaaa gaaattgttt ttgcatctcc acctaagaag gatggtaccc 3421 ttaccccaga agtcatctac gttgcccaca aggttggtgc taagtgtatc gtgctagcag 3481 gaggcgccca ggcagtagct gctatggctt acggaacaga aactgttcct aagtgtgaca 3541 aaatatttgg tccaggaaac cagttcgtta ctgctgccaa gatgatggtt caaaatgaca 3601 catcagccct gtgtagtatt gacatgcctg ctgggccttc tgaagttcta gttattgctg 3661 ataaatacgc tgatccagat ttcgttgcct cagaccttct gtctcaagct gaacatggta 3721 ttgattccca ggtgattctg ttggctgtcg atatgacaga caaggagctt gccagaattg 3781 aagatgctgt tcacaaccaa gctgtgcagt tgccaagggt tgaaattgta cgcaagtgta 3841 ttgcacactc tacaacccta tcggttgcaa cctacgagca ggctttggaa atgtccaatc 3901 agtacgctcc tgaacacttg atcctgcaaa tcgagaatgc ttcttcttat gttgatcaag 3961 tacaacacgc tggatctgtg tttgttggtg cctactctcc agagagttgt ggagattact 4021 cctccggtac caaccacact ttgccaacgt acggatatgc ccgtcaatac agcggagtta 4081 acactgcaac cttccagaag ttcatcactt cacaagacgt aactcctgag ggactgaaac 4141 atattggcca agcagtgatg gatctggctg ctgttgaagg tctagatgct caccgcaatg 4201 ctgttaaggt tcgtatggag aaactgggac ttatttaatt atttagagat tttaacttac 4261 atttagattc gatagatcca caggacgggt gtggtcgcca tgatcgcgta gtcgatagtg 4321 gctccaagta gcgaagcgag caggactggg cggcggccaa agcggtcgga cagtgctccg 4381 agaacgggtg cgcatagaaa ttgcatcaac gcatatagcg ctagcagcac gccatagtga 4441 ctggcgatgc tgtcggaatg gacgatatcc cgcaagaggc ccggcagtac cggcataacc 4501 aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta 4561 gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa 4621 agcttatcga taagcttgca caaacgaacg cttttctttg ggcaaggaaa aatcaagaaa 4681 aagcagaggt taaagttttc aggggaatgg caattgcttt atatatggga gaaagttaac 4741 tacgtcggtg ctgtaggcgt agagagcgac tggagaatgc gtgatgaggt cgtctctttt 4801 cgccccccct tggcggggta aaaattgcac tactgcagaa ttactacacc cctattccga 4861 ggagacggag tgcgacaaaa atggtaaagt tcaccctagt ctgcgacttt taattgacgg 4921 acaccggcgt ttacatgcga aaaaaactaa agtgcgcgca tttcacggcc gaggggggtc 4981 ccacttggga ctgagagggg gtgggatctg aaatcgagga ggtatcaaga ccccccgttt 5041 ctcaactccc taatcaaaaa ttacgaagtc ctcgttggaa aggagttaaa ataattaagc 5101 ggggtcggac gccataccga ggttatcttg caggcatttt actaatattg gaattcggag 5161 ctcaacttgc aaccaggcag ggtttagcta tgtaatcaat gtaatcaata taataaagca 5221 ctaccacatc gaaggtttgg gagggaggcc aatagtgtcc cccacagggt gctgatatcg 5281 cgattcttgg gtgaggagac acatatttca ctcctctcac caaccaacca agcggctcct 5341 cgcaagatga tttatccgat tatccggaca ctatactccc atccagtttg atgccgattt 5401 catcgattgt cctaaataat ccttaaatat gtatagaacg gtaccctggg gttacataat 5461 ccttatttaa taatccctcc cccaccgctt ttcttttttt tttcttctta ttgtcttagc 5521 ctcgggtact ccgcaagagg ttgacgaagt cttcctccaa gtcgtcgttt ccattccttg 5581 acgtcgtggt attaccattg gtattgtgat gtagaaattc ctgtggattt gcgtagttaa 5641 actgctggtc tgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 5701 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 5761 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 5821 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 5881 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 5941 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 6001 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 6061 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 6121 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 6181 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 6241 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 6301 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 6361 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 6421 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 6481 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 6541 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 6601 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 6661 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 6721 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 6781 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 6841 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 6901 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 6961 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 7021 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 7081 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 7141 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 7201 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 7261 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 7321 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 7381 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 7441 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 7501 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 7561 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 7621 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 7681 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 7741 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 7801 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 7861 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 7921 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 7981 cgtcagggcg cgtcagcggg tgttggc //