LOCUS Exported 4568 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION EasyClone-MarkerFree backbone vector for the addition of 1 or 2 genes and promoters for integration into site XI-2 (Chr XI: 91575..92913). ACCESSION . VERSION . KEYWORDS pCfB2903(XI-2 MarkerFree) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4568) AUTHORS Jessop-Fabre MM, Jakociunas T, Stovicek V, Dai Z, Jensen MK, Keasling JD, Borodina I TITLE EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. JOURNAL Biotechnol J. 2016 Aug;11(8):1110-7. doi: 10.1002/biot.201600147. Epub 2016 Jun 23. PUBMED 27166612 REFERENCE 2 (bases 1 to 4568) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4568 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 622..641 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(860..951) /gene="bla" /label=AmpR promoter primer_bind 993..1012 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 1018..1037 /label=pENTR-R /note="pENTR vectors, reverse primer" protein_bind 1244..1277 /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)" terminator 1925..2090 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind 2011..2035 /label=pHybLex-R /note="ADH terminator, reverse primer" terminator 2149..2338 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(2355..2374) /label=T7 /note="T7 promoter, forward primer" promoter complement(2356..2374) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2384..2400) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2393..2415) /label=M13/pUC Forward /note="In lacZ gene" protein_bind complement(3066..3099) /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(3569..3585) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3684..3701) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3855..4443) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3935..3954) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4567..859) /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" ORIGIN 1 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 61 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 121 gcgctgcgat gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc 181 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 241 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 301 ttgttgccat cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 361 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 421 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 481 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 541 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 601 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 661 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 721 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 781 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 841 ggaaatgttg aatactcata ttcttccttt ttcaatatta ttgaagcatt tatcagggtt 901 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggtca 961 gtgttacaac caattaacca attctgaaca ttatcgcgag cccatttata cctgaatatg 1021 gctcataaca ccccttgttt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg 1081 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggactcc ccatgcgaga 1141 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg 1201 cccgggctaa ttatggggtg tcgcccttat tcgactctat agtgaagttc ctattctcta 1261 gaaagtatag gaacttctga agtggggatt taaatgcggc cgcgctgagg gtttaatgag 1321 gattttcgat ggagcaggat gaggagaaat agtaccacat gtatatatcc attacaaaaa 1381 ggtttatata caattacaat agacccttgt tggggtttct gaaaaaagaa gtagtcgatg 1441 ccatcggcaa taatacggaa ttacgagaaa cacaatcccg atcctttttt gggtaattac 1501 ttcaccgatt ctaccgattt atcatgccaa aaaaaattca ccgtgggttc tagaagtgcc 1561 ctttgaggat tgtagccact ctaacccaca cggcctcctt actagctgac taaggtgaca 1621 aaaccgcaag gactggaaag tcgccactca tctgaaaatt ctcaagtttt tcactactga 1681 gtttatgctt tcgaattttt ttgttcggta atagcacggc ggttcgattc aattccgccg 1741 ctccgagcga tgctccgcaa aactcagtaa taagctttct gatggttcac ccctttttta 1801 gcacgcgggg tgtaactcaa cagaaaaatg tgccatagaa caagactagg caaaagccaa 1861 ggagcgtttg ccatgaactt ccacaaatta agtcctcagc gagctcgcat ggaatgcgtg 1921 cgatgagcga cctcatgcta tacctgagaa agcaacctga cctacaggaa agagttactc 1981 aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta tacacttatt 2041 ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc ttatttagaa 2101 gtgtcaacaa cgtatctacc aacggaatgc gtgcgatcgc gtgcattcat ccgctctaac 2161 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat 2221 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt 2281 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaagat 2341 cgcgacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac 2401 aacgtcgtga ctgggaaaac cctggcgtta cccctgcagg actagtgctg aggcattaat 2461 cacacgacta gcgctttcag atattaaaaa gtttagatgt aggttttagc ggtaacagtt 2521 atataaatcg tgtttcttct cttgatgaaa caaaaaaatg ctagaaaaac tttgtcgttt 2581 cttacttttg gtgcgctttg cagttttcgt ggctagactt agaatcattt ctcctcagat 2641 ttcttgatta aagtttggtg cgaagcccta ctctaacatt ggtgttcttc ttttcattca 2701 cgcaagttaa gtccaggaag gtgagcaaat gctcatcctt ctgttcatgc gtgacggctg 2761 aattatcctt atctggcgta cccgtgcagc cgtttccgtg cctcggttcc tccgagatat 2821 ccttagggac cgccagggac catgattgcg tcaactgttg tcaccgctcc agaggatcct 2881 ctgtaacctt ttcaaccata aaaatagaaa gggcacaggg ttggggtatt tgaatttttt 2941 ttttgggttt tttcgtatta cttattactt accttctttt ctatataatt ttgtttttcc 3001 ctggtagagc ggaatcttcc cacattaaga cctcagcgcg gccgcaaatt taaataaaat 3061 gaagtgaagt tcctatactt tctagagaat aggaacttct atagtgagtc gaataagggc 3121 gacacaaaat ttattctaaa tgcataataa atactgataa catcttatag tttgtattat 3181 attttgtatt atcgttgaca tgtataattt tgatatcaaa aactgatttt ccctttatta 3241 ttttcgagat ttattttctt aattctcttt aacaaactag aaatattgta tatacaaaaa 3301 atcataaata atagatgaat agtttaatta taggtgttca tcaatcgaaa aagcaacgta 3361 tcttatttaa agtgcgttgc ttttttctca tttataaggt taaataattc tcatatatca 3421 agcaaagtga caggcgccct taaatattct gacaaatgct ctttccctaa actcccccca 3481 taaaaaaacc cgccgaagcg ggtttttacg ttatttgcgg attaacgatt actcgttatc 3541 agaaccgccc agggggcccg agcttaagac tggccgtcgt tttacaacac agaaagagtt 3601 tgtagaaacg caaaaaggcc atccgtcagg ggccttctgc ttagtttgat gcctggcagt 3661 tccctactct cgccttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 3721 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 3781 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 3841 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 3901 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 3961 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 4021 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 4081 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 4141 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4201 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 4261 ttgaagtggt gggctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 4321 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 4381 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 4441 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga cgcgcgcgta 4501 actcacgtta agggattttg gtcatgagct tgcgccgtcc cgtcaagtca gcgtaatgct 4561 ctgctttt //