LOCUS Exported 5146 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION EasyCloneYALI system-based yeast integrative vector for markerfree integration via CRISPR/Cas9 to be used in combination with pCfB6627, integration into Yarrowia lipolytica chromosomal location IntC_2, USER site for cloning, amp resistance. ACCESSION . VERSION . KEYWORDS pCfB6682 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5146) 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 5146) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5146 /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 complement(2443..2476) /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(2946..2963) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2946..2962) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3061..3078) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3232..3820) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3312..3331) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3942..4802) /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 4565..4584 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4803..4894) /gene="bla" /label=AmpR promoter primer_bind 4936..4955 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4961..4980 /label=pENTR-R /note="pENTR vectors, reverse primer" ORIGIN 1 gggctaatta tggggtgtcg cccttattcg actctatagt gaagttccta ttctctagaa 61 agtataggaa cttctgaagt ggggatttaa atgcggccgc gaatgcacgc gatgattgaa 121 acgcctgaca aaaacgcctg ataaaaacgc ctgataaaaa cgcctgataa aaacgcctga 181 taaaaacgcc tgtcaaaacc aaccagacct cggtagagag gatcagagtc ccatggtgta 241 ctatacagtg aaactgctgt gcgaatggta taccattgct gtctatatag cctctagagt 301 ggcactaatc cacaaggaca cagcacaatg tactacaggg tagtactcac cccacagatg 361 ctcttcgatg aaatagacag agaatgactg ggccagtacc attgccaact tgtagggaat 421 cgtcactctc caattccagc tccagttcca gttccagctc tttcggctta attctagctc 481 tcatttcggc atcttcattc agctgttcag gttcctgaca ctgctcaatt cggcgatctc 541 gacgactgca tcctgacttc agtctcgacc ctataattgt gggctgcaca ctcggaggta 601 taggccactc agatgcattc ttgggcggtc tataccagct cattgaccct tcgtgggtct 661 caatgttcaa cgcagatgaa cttcaaatgc ttatcagtgg aggacatgcc aatgtggacg 721 tctgggattt gaagacacac accaattacg gaggctatct ggatacagat caaactataa 781 aggacttctg gactgtgttt gagagtttcg aggaagagga caagcggctg cttcttaagt 841 ttgtgacatc agtatccaag gcaccattgc aaggattcaa ggctttgaac ccgtcatttg 901 ccattcgtaa cgctggtaga caggttgatc ggttccctac ggcctccacc tgtgtcaatc 961 ttctcaagct gcctgactat caggacattg atcaacttcg gaagaaactt ttgtatgcca 1021 ttcgatcgca tgctggtttc gatttgtctt agaggaacgc atatacagta atcatagaga 1081 ataaacgata ttcatttatt aaagtagata gttgaggtag aagttgtaaa gagtgataaa 1141 tagcttagat accacagaca ccctcggtga cgaagtactg cagatggttt ccaatcacat 1201 tgacctgctg gagcagagtg ttaccggcag agcactgttt attgctctgg ccctggcaca 1261 tgacaacgtt ggagagagga gggtggatca ggggccagtc aataaagacc tcaccagagc 1321 agtgctggta accgtcccag aagggcactt gagggacgat atctcctcgg tgggtgattc 1381 ggtagagctt tcggtctttg gacaccttgg agacatcggg gttctcctgg ccaaagaaga 1441 gtttatcgac ccagttagca aagccagcgt taccgacaat gggctgacca agagtaacaa 1501 cgaggggatc gtggccgtta accttgaggt tgattccgaa cagaaggaat gcacgcgatc 1561 gcacgcattc cgttgaagtg tggatgggga agtgagtgcc cggttctgtg tgcacaattg 1621 gcaatccaag atggatggat tcaacacagg gatatagcga gctacgtggt ggtgcgagga 1681 tatagcaacg gatatttatg tttgacactt gagaatgtac gatacaagca ttgtccaagt 1741 acaatactaa acatactgta catactcata ctcgtacccg ggcaacggtt tcacttgagt 1801 gcagtggcta gtgcccttac tcgtacagtg tgcaatactg cgtatcatag tctttgatgt 1861 atatcgtatt cattcatgtt agttgcgtaa gcgttgcacg tttccatcta agacctacat 1921 ttgtctgtta gctttgtgta gtcttagacg gcttgtagca gtccggagcg agattgtgtt 1981 tctcgttgac cataagctga acactgctgt ttccagcctc agaagcgcag tcctcgtatc 2041 gtttctgctg ctgcggttgt tgttgttgtt gttgttgttg ttgttgttgg atgtcgtcct 2101 tcgcgtctct ggcccatcac caaggtggaa ggtggatctt cgtcttcacg aagttggtac 2161 ttgttggcaa acatctcatt gaccatatcc cacatcaccc tggggcctgt gttcgaagga 2221 tagagcgggt gcgcaaccac ccagagaagc attcaaaaca atacctacaa gttgatctct 2281 ttcggtgtct ccagataccc atctccacaa gtgagctttc tccacgaatc gtccattgac 2341 acctgtccag aaacaccaac cggtatttca atgagtcgcc gagtgttcag gtcgtcgaat 2401 tatcgcacgc attcgcggcc gcaaatttaa ataaaatgaa gtgaagttcc tatactttct 2461 agagaatagg aacttctata gtgagtcgaa taagggcgac acaaaattta ttctaaatgc 2521 ataataaata ctgataacat cttatagttt gtattatatt ttgtattatc gttgacatgt 2581 ataattttga tatcaaaaac tgattttccc tttattattt tcgagattta ttttcttaat 2641 tctctttaac aaactagaaa tattgtatat acaaaaaatc ataaataata gatgaatagt 2701 ttaattatag gtgttcatca atcgaaaaag caacgtatct tatttaaagt gcgttgcttt 2761 tttctcattt ataaggttaa ataattctca tatatcaagc aaagtgacag gcgcccttaa 2821 atattctgac aaatgctctt tccctaaact ccccccataa aaaaacccgc cgaagcgggt 2881 ttttacgtta tttgcggatt aacgattact cgttatcaga accgcccagg gggcccgagc 2941 ttaagactgg ccgtcgtttt acaacacaga aagagtttgt agaaacgcaa aaaggccatc 3001 cgtcaggggc cttctgctta gtttgatgcc tggcagttcc ctactctcgc cttccgcttc 3061 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 3121 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 3181 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 3241 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 3301 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 3361 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 3421 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 3481 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 3541 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 3601 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggg ctaactacgg 3661 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 3721 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 3781 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 3841 tacggggtct gacgctcagt ggaacgacgc gcgcgtaact cacgttaagg gattttggtc 3901 atgagcttgc gccgtcccgt caagtcagcg taatgctctg cttaccaatg cttaatcagt 3961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 4021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc gatgataccg 4081 cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc cggaagggcc 4141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 4201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc catcgctaca 4261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 4321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 4381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 4441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 4501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 4561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 4621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 4681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 4741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 4801 atattcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 4861 tacatatttg aatgtattta gaaaaataaa caaatagggg tcagtgttac aaccaattaa 4921 ccaattctga acattatcgc gagcccattt atacctgaat atggctcata acaccccttg 4981 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa 5041 acgccgtagc gccgatggta gtgtggggac tccccatgcg agagtaggga actgccaggc 5101 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgccc //