LOCUS Exported 5651 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Part of GoldenGate Yarrowia lipolytica toolkit for assembling an expression plasmid for Y. lipolytic yeast. Metabolic marker for selection after transformation in Y. lipolytica (Marker_Suc2). ACCESSION . VERSION . KEYWORDS GGE142 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5651) AUTHORS Larroude M, Park YK, Soudier P, Kubiak M, Nicaud JM, Rossignol T TITLE A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology. JOURNAL Microb Biotechnol. 2019 May 31. doi: 10.1111/1751-7915.13427. PUBMED 31148366 REFERENCE 2 (bases 1 to 5651) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5651 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /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 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 239..257 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 239..256 /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2539..2558) /label=T7 /note="T7 promoter, forward primer" promoter complement(2540..2558) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2565..2582) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2565..2581) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2574..2596) /label=M13/pUC Forward /note="In lacZ gene" CDS 2719..3021 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 2900..2919 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 3370..4164 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3424..3443) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4034..4053 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 4364..4373 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4370..4744 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" rep_origin 4882..5470 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5371..5390 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5624..5641 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat 241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca 301 ctagtaacgg ccgccagtgt gctggaattc gcccttgggg gtctctccgt gaattcgatt 361 tgtcttagag gaacgcatat acagtaatca tagagaataa acgatattca tttattaaag 421 tagatagttg aggtagaagt tgtaaagagt gataaatagc ttagatacca cagacaccct 481 aggctgccta ttttacttcc cttacttgga acttgtcaat gtagaacaaa ttatcgacac 541 cagtggtcat gttcacagat cctagagcgt taccggtggt catgaagtag gtatttgtag 601 aaaccacatc tccatcgttg aagtacaatt ccaagatgtt ttgatccagt aggccgtaca 661 ctttatagta acttaggtcg ttctcagact tgaatggttg gttgttgaca gacattctgt 721 ttgtgaaata tgggttctcc ttgacaaact tgaccttaga gttaccacgg tccaaaaaga 781 aggaagaagc actgacttca aaacccattc tcaaatattc ttcaggatct tctaaaccct 841 tgaaccaaag tgataagtcg gcaaagacgg atttggatat ggtttgtgtg gtgttaacag 901 cgtaaaccaa ctcaaactct agggtaccag tcgagttgct caaatcgaca ttgtaagaat 961 tggccttagt tagagttgtg ttagtagcaa aacgggacca gggaccagca ttactaatgt 1021 tcaatattgg ttcggctttc aaattgatca attcagtctc tggattagct tgatattcag 1081 tgttcaaaga aaacttgcgg accaaagaca tggatgatct ccatgggtta gttgggacaa 1141 aggcactgta ctcccagttt gaagcccagg caatacctaa tgctgaaccg taggttgggt 1201 cagtgttgaa gaaagtttgc aaggcatagt agtccttacc aaaatctacc actctagatt 1261 gattgtcaaa cgcttcaaaa tgagtaccat tgaaggatcc aacaaaatat tggttgaagg 1321 aaccgccagc aggtgcacct gggttgatag aaataaacat gacccaataa gatttggaag 1381 gatcttgctc agttgggact tcaatcaaac ctggacattc gtattggtag cctaagaaac 1441 cttcattggc aaatgcagat tctagcttcc aggacttcaa gtcatcagag gagtaaattt 1501 caattttgta gtcttgtgat ttggcagccg tcataatcca tttttgagaa ggttcatacc 1561 agaacacctt tggatctctg aattgagtgg agttggcagc taaaacaggg ttcttttggt 1621 attcagtaaa agtgtaacca ccatcaagag aatagctaat gtattgctct tcactttcag 1681 gagtgttata agtccaaatc gcaacgcatc tttgtcttgg atcaatagta tcattgaaaa 1741 acccactcgt gttgttgtaa tcaaccacca tggagccaga gaaagcacct gaatcgttac 1801 gcttgggagc gatagcaatg ggttgatctt cccaattagt caaatcatcg gaagtagcat 1861 ggccccaaaa caatggcgta ccccatacgg tgtcatttgg gttgtattga aagtacagat 1921 gccatttggc atctttttca tcgtaccaca acccatttgg gtcattcatc cagcccttgt 1981 tgggtgtgaa gtggaccaaa ggtctatcgc tagtttcgtt tgtcattgat gcagatattt 2041 tggctgcaaa accagccaaa aggaaaagga aagcttgcaa aagcattgtg agatccttcg 2101 ggtgtgagtt gacaaggaga gagagaaaag aagaggaaag gtaattcggg gacggtggtc 2161 ttttataccc ttggctaaag tcccaaccac aaagcaaaaa aattttcagt agtctatttt 2221 gcgtccggca tgggttaccc ggatggccag acaaagaaac tagtacaaag tctgaacaag 2281 cgtagattcc agactgcagt accctacgcc cttaacggca agtgtgggaa ccgggggagg 2341 tttgatatgt ggggtgaagg gggctctcgc cggggttggg tccgctactg ggtcaatttg 2401 gggtcaattg gggcaattgg ggctgttttt tgggacacaa atgcgccgcc aacccaccta 2461 gagaccccca agggcgaatt ctgcagatat ccatcacact ggcggccgct cgagcatgca 2521 tctagagggc ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta 2581 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 2641 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 2701 cgcagcctat acgtacggca gtttaaggtt tacacctata aaagagagag ccgttatcgt 2761 ctgtttgtgg atgtacagag tgatattatt gacacgccgg ggcgacggat ggtgatcccc 2821 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat 2881 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt 2941 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac 3001 ctgatgttct ggggaatata aatgtcaggc atgagattat caaaaaggat cttcacctag 3061 atccttttca cgtagaaagc cagtccgcag aaacggtgct gaccccggat gaatgtcagc 3121 tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt 3181 gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga accggaattg 3241 ccagctgggg cgccctctgg taaggttggg aagccctgca aagtaaactg gatggctttc 3301 ttgccgccaa ggatctgatg gcgcagggga tcaagctctg atcaagagac aggatgagga 3361 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag 3421 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc 3481 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg 3541 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 3601 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg 3661 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct 3721 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg 3781 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat 3841 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc 3901 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg 3961 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc 4021 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct 4081 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat 4141 cgccttcttg acgagttctt ctgaattatt aacgcttaca atttcctgat gcggtatttt 4201 ctccttacgc atctgtgcgg tatttcacac cgcatcaggt ggcacttttc ggggaaatgt 4261 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 4321 acaataaccc tgataaatgc ttcaataata gcacgtgagg agggccacca tggccaagtt 4381 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4441 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4501 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4561 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 4621 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 4681 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 4741 ctgacacgtg ctaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 4801 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 4861 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 4921 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 4981 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 5041 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 5101 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 5161 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 5221 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 5281 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 5341 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 5401 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 5461 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 5521 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 5581 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 5641 ggaagcggaa g //