LOCUS Exported 6599 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION EasyCloneYALI system-based yeast integrative vector carrying loxP-flanked nourseothricin-resistance marker, integration into Yarrowia lipolytica chromosomal location IntE_1, USER site for cloning, amp resistance. ACCESSION . VERSION . KEYWORDS pCfB4666 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6599) AUTHORS Holkenbrink C, Dam MI, Kildegaard KR, Beder J, Dahlin J, Domenech Belda D, Borodina I TITLE EasyCloneYALI: CRISPR/Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica. JOURNAL Biotechnol J. 2018 Jan 29. doi: 10.1002/biot.201700543. PUBMED 29377615 REFERENCE 2 (bases 1 to 6599) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6599 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 41..74 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" protein_bind 639..672 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 871..889 /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" protein_bind 2007..2040 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(3896..3929) /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" primer_bind complement(4399..4416) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4399..4415) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4514..4531) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4685..5273) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4765..4784) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5395..6255) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6018..6037 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6256..6347) /gene="bla" /label=AmpR promoter primer_bind 6389..6408 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 6414..6433 /label=pENTR-R /note="pENTR vectors, reverse primer" ORIGIN 1 gggctaatta tggggtgtcg cccttattcg actctatagt gaagttccta ttctctagaa 61 agtataggaa cttctgaagt ggggatttaa atgcggccgc gaatgcacgc gatgttagaa 121 gcaattggag aagaaacgtt cagtaacttc aaaactagtt ttcctctgtc aaattttgga 181 tcagatcaag tgtcctatca gtctcgccct tgtgtttgca cggtctgcca agttctctca 241 gatgatctac gttcaactaa tgagacagaa agagcacgca ggcttgaaag caggcttgaa 301 cgtatttggt tgagtcattg tacggcaact ggaaggggct gagttgatct atctgtcttc 361 ttcttcctct tcttcttcaa atgacatcag cactggagat tctctcctag ctcatggact 421 gagactacta tgttctatgg agttgtgctc tcaatccaac catctttcac aacatcaaac 481 tcaagtccac tgagaacatc tcgctcgact tgtctctatc tcaacaccca gggctatctg 541 agctggatcg tggaggactg gctgttgtat ttcgacacaa ccacccgagg gctgtatgct 601 tgtcgacgct cggaggccac tagtggatct gatatcacat aacttcgtat agcatacatt 661 atacgaagtt atcgagcgtc ccaaaacctt ctcaagcaag gttttcagta taatgttaca 721 tgcgtacacg cgtctgtaca gaaaaaaaag aaaaatttga aatataaata acgttcttaa 781 tactaacata actataaaaa aataaatagg gacctagact tcaggttgtc taactccttc 841 cttttcggtt agagcggatg tgggggaagg gcgtgaatgt aagcgtgaca taactaatta 901 catgatcatg gacatggcat agacatatac aaggcttgtt caccatctga tgcagtacca 961 tcgtataatg cagtatccaa accacacaag gtaaaaccca ttcttctata ggcatgaata 1021 gctggagcgt taacattagt aacttccaac cacaaatgac cagcacctct ttctctagca 1081 aattcagtag ccaaacccat caaagctcta ccaacaccat gacctctatg ttctggagca 1141 acttcaatat cttcgacggt caatcttcta ttccaaccag aataagaaac aacaacaaaa 1201 ccagccaagt caccgtcatc accataagca acaaaagttc tagaatctgg atcaccatct 1261 tcaccatcat cagattcgtc atcggattca tcatctggaa aaaccttagt caatggtgga 1321 tcaactggaa cttctctcaa agtgaaacca tcaccagtag cagtaactct gaaaacagtg 1381 tcagtagtga aagaaccatc caaagcttca atagcttcag catcacctgg aacagaagtt 1441 ctgtatctgt aagcagtatc atccaaagta gtaccctgcg gttagtactg caaaaagtgc 1501 tggtcggatg acgtggcgtc ttgtgtcgat gggaatggga ccacacccgg ccgtgtgctc 1561 aaagcccgtg gcaattgctg ctgcctctga aactcaccat tttgaatgat tcttatactc 1621 agaaggaaat gcttaacgat ttcgggtgtg agttgacaag gagagagaga aaagaagagg 1681 aaaggtaatt cggggacggt ggtcttttat acccttggct aaagtcccaa ccacaaagca 1741 aaaaaatttt cagtagtcta ttttgcgtcc ggcatgggtt acccggatgg ccagacaaag 1801 aaactagtac aaagtctgaa caagcgtaga ttccagactg cagtacccta cgcccttaac 1861 ggcaagtgtg ggaaccgggg gaggtttgat atgtggggag aagggggctc tcgccggggt 1921 tgggcccgct actgggtcaa tttggggtca attggggcaa ttggggctgt tttttgggac 1981 acaaatgcgc cgccaacccg gtctctataa cttcgtatag catacattat acgaagttat 2041 cgtacgaagc ttcagctgtt ctcagatgca ttcttgggcg gtctatacca gctcattgac 2101 ccttcgtggg tctcaatgtt caacgcagat gaacttcaaa tgcttatcag tggaggacat 2161 gccaatgtgg acgtctggga tttgaagaca cacaccaatt acggaggcta tctggataca 2221 gatcaaacta taaaggactt ctggactgtg tttgagagtt tcgaggaaga ggacaagcgg 2281 ctgcttctta agtttgtgac atcagtatcc aaggcaccat tgcaaggatt caaggctttg 2341 aacccgtcat ttgccattcg taacgctggt agacaggttg atcggttccc tacggcctcc 2401 acctgtgtca atcttctcaa gctgcctgac tatcaggaca ttgatcaact tcggaagaaa 2461 cttttgtatg ccattcgatc gcatgctggt ttcgatttgt cttagaggaa cgcatataca 2521 gtaatcatag agaataaacg atattcattt attaaagtag atagttgagg tagaagttgt 2581 aaagagtgat aaatagctta gataccacag acaccctcgg tgacgaagta ctgcagatgg 2641 tttccaatca cattgacctg ctggagcaga gtgttaccgg cagagcactg tttattgctc 2701 tggccctggc acatgacaac gttggagaga ggagggtgga tcaggggcca gtcaataaag 2761 acctcaccag agcagtgctg gtaaccgtcc cagaagggca cttgagggac gatatctcct 2821 cggtgggtga ttcggtagag ctttcggtct ttggacacct tggagacatc ggggttctcc 2881 tggccaaaga agagtttatc gacccagtta gcaaagccag cgttaccgac aatgggctga 2941 ccaagagtaa caacgagggg atcgtggccg ttaaccttga ggttgattcc gaacagaagg 3001 aatgcacgcg atcgcacgca ttccgttgaa gtgtggatgg ggaagtgagt gcccggttct 3061 gtgtgcacaa ttggcaatcc aagatggatg gattcaacac agggatatag cgagctacgt 3121 ggtggtgcga ggatatagca acggatattt atgtttgaca cttgagaatg tacgatacaa 3181 gcattgtcca agtacaatac taaacatact gtacatactc atactcgtac ccgggcaacg 3241 gtttcacttg agtgcagtgg ctagtgccct tactcgtaca gtgtgcaata ctgcgtatca 3301 tagtctttga tgtatatcgt attcattcat gttagttgcg taagcgttgc acgtaagcac 3361 tatcctctgc tgcgtcgaac tacacgcgaa agtccactaa tggcttttct attgccagcg 3421 ttctgctaga tacaggagga gggtttctgt caatgggaca gctggttttc gacggtatct 3481 tcgaccacga caggtcaggg tatctacaag aatgtgccca agtttttgct ttcctgggtc 3541 accatcttct tcgacgtggt tctcatgctc ccgcacgact ggttcccagg agctacagta 3601 cccacatcca agatggtcaa gatgtgagtg aagagcctgt gtataaggca aggggagcac 3661 tggcgtattt tgaatagatt gagtagcatg aagagcatag acgggttggc ccagtttctc 3721 tcgttagcct tctgttgcaa gtgaacagcg cacgatcttc catgttccgg tgcacaagta 3781 gacaccctag acacgccgaa aatatctccg tctcaatcaa tgggccttgg tttatcattg 3841 ttacaccata tcaaatcgca cgcattcgcg gccgcaaatt taaataaaat gaagtgaagt 3901 tcctatactt tctagagaat aggaacttct atagtgagtc gaataagggc gacacaaaat 3961 ttattctaaa tgcataataa atactgataa catcttatag tttgtattat attttgtatt 4021 atcgttgaca tgtataattt tgatatcaaa aactgatttt ccctttatta ttttcgagat 4081 ttattttctt aattctcttt aacaaactag aaatattgta tatacaaaaa atcataaata 4141 atagatgaat agtttaatta taggtgttca tcaatcgaaa aagcaacgta tcttatttaa 4201 agtgcgttgc ttttttctca tttataaggt taaataattc tcatatatca agcaaagtga 4261 caggcgccct taaatattct gacaaatgct ctttccctaa actcccccca taaaaaaacc 4321 cgccgaagcg ggtttttacg ttatttgcgg attaacgatt actcgttatc agaaccgccc 4381 agggggcccg agcttaagac tggccgtcgt tttacaacac agaaagagtt tgtagaaacg 4441 caaaaaggcc atccgtcagg ggccttctgc ttagtttgat gcctggcagt tccctactct 4501 cgccttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 4561 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 4621 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 4681 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 4741 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 4801 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 4861 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 4921 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 4981 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5041 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5101 gggctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 5161 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5221 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5281 ctttgatctt ttctacgggg tctgacgctc agtggaacga cgcgcgcgta actcacgtta 5341 agggattttg gtcatgagct tgcgccgtcc cgtcaagtca gcgtaatgct ctgcttacca 5401 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 5461 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagcgc 5521 tgcgatgata ccgcgagaac cacgctcacc ggctccggat ttatcagcaa taaaccagcc 5581 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 5641 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 5701 tgccatcgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 5761 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 5821 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 5881 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 5941 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 6001 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 6061 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 6121 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 6181 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 6241 atgttgaata ctcatattct tcctttttca atattattga agcatttatc agggttattg 6301 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggtcagtgt 6361 tacaaccaat taaccaattc tgaacattat cgcgagccca tttatacctg aatatggctc 6421 ataacacccc ttgtttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga 6481 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gactccccat gcgagagtag 6541 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgccc //