LOCUS Exported 4516 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Part of GoldenGate Yarrowia lipolytica toolkit for assembling an expression plasmid for Y. lipolytic yeast. Promoter for gene expression in position 2 (P2_GAPdh). ACCESSION . VERSION . KEYWORDS GGE005 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4516) 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 4516) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4516 /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(1404..1423) /label=T7 /note="T7 promoter, forward primer" promoter complement(1405..1423) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1430..1447) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1430..1446) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1439..1461) /label=M13/pUC Forward /note="In lacZ gene" CDS 1584..1886 /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 1765..1784 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 2235..3029 /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(2289..2308) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2899..2918 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 3229..3238 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3235..3609 /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 3747..4335 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4236..4255 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4489..4506 /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 gcccttggtc tctttgttgt tgatgtgtgt 361 ttaattcaag aatgaatata gagaagagaa gaagaaaaaa gattcaattg agccggcgat 421 gcagaccctt atataaatgt tgccttggac agacggagca agcccgccca aacctacgtt 481 cggtataata tgttaagctt tttaacacaa aggtttggct tggggtaacc tgatgtggtg 541 caaaagaccg ggcgttggcg agccattgcg cgggcgaatg gggccgtgac tcgtctcaaa 601 ttcgagggcg tgcctcaatt cgtgcccccg tggctttttc ccgccgtttc cgccccgttt 661 gcaccactgc agccgcttct ttggttcgga caccttgctg cgagctaggt gccttgtgct 721 acttaaaaag tggcctccca acaccaacat gacatgagtg cgtgggccaa gacacgttgg 781 cggggtcgca gtcggctcaa tggcccggaa aaaacgctgc tggagctggt tcggacgcag 841 tccgccgcgg cgtatcgata tccgcaaggt tccatggcgc cattgccctc cgtcggcgtc 901 tatcccgcaa cctctaaata gagcgggaat ataacccaag cttctttttt tttcctttaa 961 cacgcacacc cccaactatc atgttgctgc tgctgtttga ctctactctg tggaggggtg 1021 ctcccaccca acccaaccta caggtggatc cggcgctgtg attggctgat aagtctccta 1081 tccggactaa ttctgaccaa tgggacatgc gcgcaggacc caaatgccgc aattacgtaa 1141 ccccaacgaa atgcctaccc ctctttggag cccagcggcc ccaaatcccc ccaagcagcc 1201 cggttctacc ggcttccatc tccaagcacc cctttctcca caccccacaa aaagacccgt 1261 gcaggacatc ctactgcgtc cggtttatgc tcgccggccg gtcccggctc tttccgacta 1321 ccgaagcaga gaccaagggc gaattctgca gatatccatc acactggcgg ccgctcgagc 1381 atgcatctag agggcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg 1441 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 1501 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 1561 agttgcgcag cctatacgta cggcagttta aggtttacac ctataaaaga gagagccgtt 1621 atcgtctgtt tgtggatgta cagagtgata ttattgacac gccggggcga cggatggtga 1681 tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg 1741 tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct 1801 ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca 1861 ttaacctgat gttctgggga atataaatgt caggcatgag attatcaaaa aggatcttca 1921 cctagatcct tttcacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg 1981 tcagctactg ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt 2041 gcagtgggct tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg 2101 aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg 2161 ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa gagacaggat 2221 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg 2281 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg 2341 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 2401 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc 2461 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg 2521 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca 2581 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc 2641 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg 2701 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg 2761 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 2821 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg 2881 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 2941 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 3001 tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc ctgatgcggt 3061 attttctcct tacgcatctg tgcggtattt cacaccgcat caggtggcac ttttcgggga 3121 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 3181 atgagacaat aaccctgata aatgcttcaa taatagcacg tgaggagggc caccatggcc 3241 aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc 3301 tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc 3361 cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc 3421 ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg 3481 tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg 3541 gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag 3601 caggactgac acgtgctaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 3661 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 3721 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 3781 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 3841 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta 3901 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 3961 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 4021 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 4081 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 4141 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 4201 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 4261 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 4321 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 4381 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 4441 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 4501 agcgaggaag cggaag //