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 complement(10..28) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 96..200 /gene="bla" /label=AmpR promoter CDS 201..1061 /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 complement(419..438) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1232..1820 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1721..1740 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1974..1991 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2108..2129 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2144..2174 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2182..2198 /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)." primer_bind 2187..2209 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2206..2222 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2206..2222 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS complement(3644..6178) /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(7613..7630) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(7613..7629) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7622..7644) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(7838..7857) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7957..7979 /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2221 accatgatta cagaccagca gtttaactac gcaaatccac aggaatttct acatcacaat 2281 accaatggta ataccacgac gtcaaggaat ggaaacgacg acttggagga agacttcgtc 2341 aacctcttgc ggagtacccg aggctaagac aataagaaga aaaaaaaaga aaagcggtgg 2401 gggagggatt attaaataag gattatgtaa ccccagggta ccgttctata catatttaag 2461 gattatttag gacaatcgat gaaatcggca tcaaactgga tgggagtata gtgtccggat 2521 aatcggataa atcatcttgc gaggagccgc ttggttggtt ggtgagagga gtgaaatatg 2581 tgtctcctca cccaagaatc gcgatatcag caccctgtgg gggacactat tggcctccct 2641 cccaaacctt cgatgtggta gtgctttatt atattgatta cattgattac atagctaaac 2701 cctgcctggt tgcaagttga gctccgaatt ccaatattag taaaatgcct gcaagataac 2761 ctcggtatgg cgtccgaccc cgcttaatta ttttaactcc tttccaacga ggacttcgta 2821 atttttgatt agggagttga gaaacggggg gtcttgatac ctcctcgatt tcagatccca 2881 ccccctctca gtcccaagtg ggacccccct cggccgtgaa atgcgcgcac tttagttttt 2941 ttcgcatgta aacgccggtg tccgtcaatt aaaagtcgca gactagggtg aactttacca 3001 tttttgtcgc actccgtctc ctcggaatag gggtgtagta attctgcagt agtgcaattt 3061 ttaccccgcc aagggggggc gaaaagagac gacctcatca cgcattctcc agtcgctctc 3121 tacgcctaca gcaccgacgt agttaacttt ctcccatata taaagcaatt gccattcccc 3181 tgaaaacttt aacctctgct ttttcttgat ttttccttgc ccaaagaaaa gcgttcgttt 3241 gtgcaagctt atcgataagc tttaatgcgg tagtttatca cagttaaatt gctaacgcag 3301 tcaggcaccg tgtatgaaat ctaacaatgc gctcatcgtc atcctcggca ccgtcaccct 3361 ggatgctgta ggcataggct tggttatgcc ggtactgccg ggcctcttgc gggatatcgt 3421 ccattccgac agcatcgcca gtcactatgg cgtgctgcta gcgctatatg cgttgatgca 3481 atttctatgc gcacccgttc tcggagcact gtccgaccgc tttggccgcc gcccagtcct 3541 gctcgcttcg ctacttggag ccactatcga ctacgcgatc atggcgacca cacccgtcct 3601 gtggatctat cgaatctaaa tgtaagttaa aatctctaaa taattaaata agtcccagtt 3661 tctccatacg aaccttaaca gcattgcggt gagcatctag accttcaaca gcagccagat 3721 ccatcactgc ttggccaata tgtttcagtc cctcaggagt tacgtcttgt gaagtgatga 3781 acttctggaa ggttgcagtg ttaactccgc tgtattgacg ggcatatccg tacgttggca 3841 aagtgtggtt ggtaccggag gagtaatctc cacaactctc tggagagtag gcaccaacaa 3901 acacagatcc agcgtgttgt acttgatcaa cataagaaga agcattctcg atttgcagga 3961 tcaagtgttc aggagcgtac tgattggaca tttccaaagc ctgctcgtag gttgcaaccg 4021 atagggttgt agagtgtgca atacacttgc gtacaatttc aacccttggc aactgcacag 4081 cttggttgtg aacagcatct tcaattctgg caagctcctt gtctgtcata tcgacagcca 4141 acagaatcac ctgggaatca ataccatgtt cagcttgaga cagaaggtct gaggcaacga 4201 aatctggatc agcgtattta tcagcaataa ctagaacttc agaaggccca gcaggcatgt 4261 caatactaca cagggctgat gtgtcatttt gaaccatcat cttggcagca gtaacgaact 4321 ggtttcctgg accaaatatt ttgtcacact taggaacagt ttctgttccg taagccatag 4381 cagctactgc ctgggcgcct cctgctagca cgatacactt agcaccaacc ttgtgggcaa 4441 cgtagatgac ttctggggta agggtaccat ccttcttagg tggagatgca aaaacaattt 4501 ctttgcaacc agcaactttg gcaggaacac ccagcatcag ggaagtggaa ggcagaattg 4561 cggttccacc aggaatatag aggccaactt tctcaatagg tcttgcaaaa cgagagcaga 4621 ctacaccagg gcaagtctca acttgcaacg tctccgttag ttgagcttca tggaatttcc 4681 tgacgttatc tatagagaga tcaatggctc tcttaacgtt atctggcaat tgcataagtt 4741 cctctgggaa aggagcttct aacacaggtg tcttcaaagc gactccatca aacttggcag 4801 ttagttctaa aagggctttg tcaccatttt gacgaacatt gtcgacaatt ggtttgacta 4861 attccataat ctgttccgtt ttctggatag gacgacgaag ggcatcttca atttcttgtg 4921 aggaggcctt agaaacgtca attttgcaca attcaatacg accttcagaa gggacttctt 4981 taggtttgga ttcttcttta ggttgttcct tggtgtatcc tggcttggca tctcctttcc 5041 ttctagtgac ctttagggac ttcatatcca ggtttctctc cacctcgtcc aacgtcacac 5101 cgtacttggc acatctaact aatgcaaaat aaaataagtc agcacattcc caggctatat 5161 cttccttgga tttagcttct gcaagttcat cagcttcctc cctaatttta gcgttcaaca 5221 aaacttcgtc gtcaaataac cgtttggtat aagaaccttc tggagcattg ctcttacgat 5281 cccacaaggt ggcttccatg gctctaagac cctttgattg gccaaaacag gaagtgcgtt 5341 ccaagtgaca gaaaccaaca cctgtttgtt caaccacaaa tttcaagcag tctccatcac 5401 aatccaattc gatacccagc aacttttgag ttgctccaga tgtagcacct ttataccaca 5461 aaccgtgacg acgagattgg tagactccag tttgtgtcct tatagcctcc ggaatagact 5521 ttttggacga gtacaccagg cccaacgagt aattagaaga gtcagccacc aaagtagtga 5581 atagaccatc ggggcggtca gtagtcaaag acgccaacaa aatttcactg acagggaact 5641 ttttgacatc ttcagaaagt tcgtattcag tagtcaattg ccgagcatca ataatgggga 5701 ttataccaga agcaacagtg gaagtcacat ctaccaactt tgcggtctca gaaaaagcat 5761 aaacagttct actaccgcca ttagtgaaac ttttcaaatc gcccagtgga gaagaaaaag 5821 gcacagcgat actagcatta gcgggcaagg atgcaacttt atcaaccagg gtcctataga 5881 taaccctagc gcctgggatc atcctttgga caactctttc tgccaaatct aggtccaaaa 5941 tcacttcatt gataccatta ttgtacaact tgagcaagtt gtcgatcagc tcctcaaatt 6001 ggtcctctgt aacggatgac tcaacttgca cattaacttg aagctcagtc gattgagtga 6061 acttgatcag gttgtgcagc tggtcagcag catagggaaa cacggctttt cctaccaaac 6121 tcaaggaatt atcaaactct gcaacacttg cgtatgcagg tagcaaggga aatgtcatac 6181 ttgaagtcgg acagtgagtg tagtcttgag aaattctgaa gccgtatttt tattatcagt 6241 gagtcagtca tcaggagatc ctctacgccg gacgcatcgt ggccgacctg cagggggggg 6301 gggggcgctg aggtctgcct cgtgaagaag gtgttgctga ctcataccag gcctgaatcg 6361 ccccatcatc cagccagaaa gtgagggagc cacggttgat gagagctttg ttgtaggtgg 6421 accagttggt gattttgaac ttttgctttg ccacggaacg gtctgcgttg tcgggaagat 6481 gcgtgatctg atccttcaac tcagcaaaag ttcgatttat tcaacaaagc cgccgtcccg 6541 tcaagtcagc gtaatgctct gccagtgtta caaccaatta accaattctg aactgagtat 6601 ttatgacctt atatattatt aacgttcaat gatcatcatg agtgaaaaat cttattatta 6661 gtcgtgttct tattgtaggc agccatgatt ctctctacat gacctaacgg gttgtgtaat 6721 gctctcacgc tctttactat gtcaccccta gtcattgtgc gtcttctctt tggatgtgaa 6781 gaagtgtcgt cgttttcctt tctcttttga cgactggggt cgagcgagaa tgattcattc 6841 tgtgcggcag ctgaaggatg ggaatgggtc cttcgcaacg tttcacctgt gctctcgttg 6901 agatcgttgg ccactgaaga ctgtgctggc cgtagatggt ttccaggaaa ggatcgtggc 6961 tctgtaaaat aaggtttatc tttgatgctt gagagccgga ttaaagaggt ataattactc 7021 tctggtgtac tttgtgtagt gctgttgccg tttgctgaag gtgaactctt gttggaacat 7081 tccattatgg gtgttcgcct tgataaactt gaatggtcgc tagttagtcc tacttttgtg 7141 attgcttttg gggtctccaa ttgtccgctg gaagggggat cttgttcata tccatgtaca 7201 aaggttttat cgcgttgagg tggtgaagca tcttgtttga ccagcatcga accagtcttc 7261 ttttgcgaat gagagtccga attagttgtg gcaattgcat ctccagtaga gcggctgggc 7321 tgagctttgg aaagaatgct ttgaaaaact ccccaggcct gcttccaact tgcttgggaa 7381 cgggtatgag tatctaaagg aggagtaaca gacgaggagg gagtagacaa aaactttgtc 7441 gaggacgagg agaaagactg ttgagcctga gtcgcaggag catctggtct tctctcagcg 7501 tcattagacg tttcatgccg tgggtcaact attaatatcg gtgcattaac accacctgag 7561 gggttgtctt gattgaactg acgattgatt tctgcaggca tgcaagcttg gcactggccg 7621 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 7681 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 7741 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc 7801 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 7861 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 7921 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 7981 tttcaccgtc atcaccgaaa cgcgcga //