LOCUS Exported 4061 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Part of GoldenGate Yarrowia lipolytica toolkit for assembling an expression plasmid for Y. lipolytic yeast. Inducible promoter for gene expression in position 2 (P2_EYK300-5AB). ACCESSION . VERSION . KEYWORDS GGE129 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4061) 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 4061) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4061 /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(949..968) /label=T7 /note="T7 promoter, forward primer" promoter complement(950..968) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(975..992) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(975..991) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(984..1006) /label=M13/pUC Forward /note="In lacZ gene" CDS 1129..1431 /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 1310..1329 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 1780..2574 /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(1834..1853) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2444..2463 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 2774..2783 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2780..3154 /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 3292..3880 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3781..3800 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4034..4051 /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 aggcctgaat tcgcccttgc ggtctctttg 361 tggatccagt agatgtgtaa gtgtgtagaa gtgtcgtgtg ggccaattgt ggggaaagga 421 gcttcttata gaagctgatc tgcccctctt tctgatccac gttgaccccc acgctgtttt 481 cagctttgca aaagtttggg accgcggtac gtggtggcct tgcctcgaaa taatagtctt 541 tgtccgcact agtcaggtca tcggattatg caaggctgtg gagcttaatg cggcggcttt 601 cccttttggg attccgcttc ccgctgtgga gcttaatgcg gcggctttcc cttttgggat 661 tccgcttccc gctgtggagc ttaatgcggc ggctttccct tttgggattc cgcttcccgc 721 tgtggagctt aatgcggcgg ctttcccttt tgggattccg cttcccgctg tggagcttaa 781 tgcggcggct ttcccttttg ggattccgct tccccagatt gaaacgtaca gtatagagaa 841 aagtagatgc atcgatacgc gtatcgaagc agagaccgca agggcgaatt ctgcagatat 901 ccatcacact ggcggccgct cgagcatgca tctagagggc ccaattcgcc ctatagtgag 961 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 1021 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 1081 gcccgcaccg atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt 1141 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt 1201 gacacgccgg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa 1261 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc 1321 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac 1381 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc 1441 atgagattat caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag 1501 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca 1561 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg 1621 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg 1681 aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga 1741 tcaagctctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 1801 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 1861 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 1921 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta 1981 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 2041 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 2101 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 2161 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 2221 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 2281 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc 2341 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 2401 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 2461 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 2521 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattatt 2581 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 2641 cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 2701 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 2761 gcacgtgagg agggccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg 2821 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt 2881 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca 2941 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct 3001 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat 3061 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa 3121 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctaaaacttc atttttaatt 3181 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 3241 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 3301 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 3361 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 3421 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 3481 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 3541 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 3601 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 3661 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 3721 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 3781 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 3841 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 3901 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 3961 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 4021 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g //