LOCUS Exported 4186 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_2UASxpr-TEF). ACCESSION . VERSION . KEYWORDS GGE150 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4186) 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 4186) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4186 /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(1074..1093) /label=T7 /note="T7 promoter, forward primer" promoter complement(1075..1093) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1100..1117) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1100..1116) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1109..1131) /label=M13/pUC Forward /note="In lacZ gene" CDS 1254..1556 /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 1435..1454 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 1905..2699 /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(1959..1978) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2569..2588 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 2899..2908 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2905..3279 /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 3417..4005 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3906..3925 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4159..4176 /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 tctttgtgga tccttcgggt 361 gtgagttgac aaggagagag agaaaagaag aggaaaggta attcggggac ggtggtcttt 421 tatacccttg gctaaagtcc caaccacaaa gcaaaaaaat tttcagtagt ctattttgcg 481 tccggcatgg gttacccgga tggccagaca aagaaactag tacaaagtct gaacaagcgt 541 agattccaga ctgcagtacc ctacgccctt aacggcaagt gtgggaaccg ggggaggttt 601 gatatgtggg gtgaaggggg ctctcgccgg ggttgggtcc gctactgggt caatttgggg 661 tcaattgggg caattggggc tgttttttgg gacacaaatg cgccgccaac cccctctcta 721 tcgaacgcac ttttgcccgt gatcagtgca tgccggatcg aggtgggcgg gaacaaccct 781 ctcgataatg tacgtacact acacacaaag agaagcaagt gggggcggga ctgcacggca 841 cttgtgagac acctcagcat gccggatcga ggtgggcggg aacaaccctc tcgataatgt 901 acgtacacta cacacaaaga gaagcaagtg ggggcgggac tgcacggcac ttgtgagaca 961 cctcagcatg cacgcgtatc gatacgcgta tcgaagcaga gaccaagggc gaattctgca 1021 gatatccatc acactggcgg ccgctcgagc atgcatctag agggcccaat tcgccctata 1081 gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 1141 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 1201 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctatacgta cggcagttta 1261 aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata 1321 ttattgacac gccggggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag 1381 ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga 1441 tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca 1501 gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt 1561 caggcatgag attatcaaaa aggatcttca cctagatcct tttcacgtag aaagccagtc 1621 cgcagaaacg gtgctgaccc cggatgaatg tcagctactg ggctatctgg acaagggaaa 1681 acgcaagcgc aaagagaaag caggtagctt gcagtgggct tacatggcga tagctagact 1741 gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg 1801 ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca 1861 ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 1921 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 1981 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 2041 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc 2101 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 2161 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 2221 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 2281 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 2341 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 2401 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg 2461 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 2521 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 2581 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 2641 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa 2701 ttattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt 2761 cacaccgcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 2821 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 2881 taatagcacg tgaggagggc caccatggcc aagttgacca gtgccgttcc ggtgctcacc 2941 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac 3001 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg 3061 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac 3121 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg 3181 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc 3241 ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctaaa acttcatttt 3301 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 3361 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 3421 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 3481 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 3541 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag 3601 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 3661 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 3721 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 3781 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 3841 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 3901 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 3961 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 4021 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 4081 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 4141 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaag //