LOCUS Exported 3857 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Part of GoldenGate Yarrowia lipolytica toolkit for assembling an expression plasmid for Y. lipolytic yeast. Sequence for integration into Y. lipolytica genome, randomly or at zeta platforms. Linearization of cassette by NotI restriction enzyme after the assembly. ACCESSION . VERSION . KEYWORDS GGE067 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3857) 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 3857) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3857 /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(745..764) /label=T7 /note="T7 promoter, forward primer" promoter complement(746..764) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(771..788) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(771..787) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(780..802) /label=M13/pUC Forward /note="In lacZ gene" CDS 925..1227 /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 1106..1125 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 1576..2370 /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(1630..1649) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2240..2259 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 2570..2579 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2576..2950 /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 3088..3676 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3577..3596 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3830..3847 /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 tctaccttct agcaaagtgc 361 tttgtgcgta ccagggatag ggtaggtagt gaaatctgag ttagtacatc aactctagac 421 gatgggcgtc gcctgtgtag aagaacaata actcacccgg taactaacac tatttctcgg 481 tggtcaatgc gtcagaagat atcaagacgg tccgttttgc gtttaagccg agtgaatgtt 541 gcctgccgtt agtaaattta ttatgaaaaa ccccactatg aatacatcag cctatactga 601 tataccaaga agtgcaaggg aggtggtcct gttccacctg aacgcggttc ccgacagcgg 661 ccgcaggcag agaccaaggg cgaattctgc agatatccat cacactggcg gccgctcgag 721 catgcatcta gagggcccaa ttcgccctat agtgagtcgt attacaattc actggccgtc 781 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 841 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 901 cagttgcgca gcctatacgt acggcagttt aaggtttaca cctataaaag agagagccgt 961 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgccggggcg acggatggtg 1021 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg 1081 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc 1141 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc 1201 attaacctga tgttctgggg aatataaatg tcaggcatga gattatcaaa aaggatcttc 1261 acctagatcc ttttcacgta gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat 1321 gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa gcaggtagct 1381 tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc aagcgaaccg 1441 gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg 1501 gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca agagacagga 1561 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg 1621 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc 1681 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt 1741 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt 1801 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc 1861 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc 1921 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac 1981 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag 2041 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag 2101 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat 2161 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg 2221 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa 2281 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc 2341 ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt cctgatgcgg 2401 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tcaggtggca cttttcgggg 2461 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 2521 catgagacaa taaccctgat aaatgcttca ataatagcac gtgaggaggg ccaccatggc 2581 caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt 2641 ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt 2701 ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac 2761 cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt 2821 gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg 2881 ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga 2941 gcaggactga cacgtgctaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 3001 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 3061 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 3121 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 3181 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 3241 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 3301 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 3361 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 3421 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 3481 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 3541 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 3601 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 3661 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 3721 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 3781 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 3841 gagcgaggaa gcggaag //