LOCUS Exported 4547 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-3 (Chr XI: 93378..94567). ACCESSION . VERSION . KEYWORDS pCfB2904(XI-3 MarkerFree) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4547) 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 4547) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4547 /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 1931..2096 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind 2017..2041 /label=pHybLex-R /note="ADH terminator, reverse primer" terminator 2155..2344 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(2361..2380) /label=T7 /note="T7 promoter, forward primer" promoter complement(2362..2380) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2390..2406) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2399..2421) /label=M13/pUC Forward /note="In lacZ gene" protein_bind complement(3045..3078) /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(3548..3564) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3663..3680) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3834..4422) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3914..3933) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4546..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 gtttaatagt 1321 tacttgctct atgcgtttgc gcatcctctt tttacttttt ttttttcagt aaagcctaag 1381 cataaatcgt tttatacgta cgacacgttc aacttttctt ggttagtagt ggcaatctct 1441 gcaatacata cagggagtca tggtctatca tcttgtccaa tcaaagaagc atcggttcag 1501 atcgagcaaa ctgtagggag aaaggaaagt agaaatgcag agtgtgctat atgtccaatc 1561 tcggttttgt agtttggatg tcattagaga tctaccaccc aaccggctgc tttcatgtgg 1621 aacagaaaag aaatcggggc gcttcctctt ctgtattcct ttagttaacg tttttattca 1681 gccatctaac catcataccc ccatacggta acaaaacctc ttctaagaaa agaagtctct 1741 gctcctccgc catcttattt ttattcgctg cgcgcgttta ttgtcgcatc gctggccagc 1801 aaaaaattgg ttgccttttt ttacctaaaa aagacacatc taactgatta gttttccgtt 1861 ttaggatatt gacgccaagc gtgcgtctga ttattaagtc ctcagcgagc tcgcatggaa 1921 tgcgtgcgat gagcgacctc atgctatacc tgagaaagca acctgaccta caggaaagag 1981 ttactcaaga ataagaattt tcgttttaaa acctaagagt cactttaaaa tttgtataca 2041 cttatttttt ttataactta tttaataata aaaatcataa atcataagaa attcgcttat 2101 ttagaagtgt caacaacgta tctaccaacg gaatgcgtgc gatcgcgtgc attcatccgc 2161 tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat 2221 agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag 2281 acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc 2341 gaagatcgcg tcccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg 2401 ttttacaacg tcgtgactgg gaaaaccctg gcgttacccc tgcaggacta gtgctgaggc 2461 attaatttac gtggattgag ccagcaatac agatcattat taaattgttt tgtacatgat 2521 gttagtatat aatcgtaaag cttttctaat atgtatacct tatacatgga actccacaga 2581 acttgcaaac ataccaaaaa tcccttattc ttgttcactc attttacatc aaaaaataat 2641 atttcagtta ttaaggaaaa taaaaaaata gattagagaa gcattttgaa gaaatagtat 2701 attcttttat tgaacctaag agcgtgatat ttttactcga aataaaatac gaaaaatcta 2761 tgcactcatc tttccgacta ctattggctc ctgctcaaaa aaagagggaa aaaaagctcc 2821 aaaattctat cttttcctat cgctcctgtc ctatccttat tacgttcatt actattttaa 2881 tactatccat tcttttattt tcagtctaaa aaaaacattt ctcataacgg gaaaagcaaa 2941 aaaatgtcaa gcttatacat caaaacacca ctgcatgcat tatctgctgg tccggattct 3001 caattaagac ctcagcgcgg ccgcaaattt aaataaaatg aagtgaagtt cctatacttt 3061 ctagagaata ggaacttcta tagtgagtcg aataagggcg acacaaaatt tattctaaat 3121 gcataataaa tactgataac atcttatagt ttgtattata ttttgtatta tcgttgacat 3181 gtataatttt gatatcaaaa actgattttc cctttattat tttcgagatt tattttctta 3241 attctcttta acaaactaga aatattgtat atacaaaaaa tcataaataa tagatgaata 3301 gtttaattat aggtgttcat caatcgaaaa agcaacgtat cttatttaaa gtgcgttgct 3361 tttttctcat ttataaggtt aaataattct catatatcaa gcaaagtgac aggcgccctt 3421 aaatattctg acaaatgctc tttccctaaa ctccccccat aaaaaaaccc gccgaagcgg 3481 gtttttacgt tatttgcgga ttaacgatta ctcgttatca gaaccgccca gggggcccga 3541 gcttaagact ggccgtcgtt ttacaacaca gaaagagttt gtagaaacgc aaaaaggcca 3601 tccgtcaggg gccttctgct tagtttgatg cctggcagtt ccctactctc gccttccgct 3661 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 3721 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 3781 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 3841 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 3901 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 3961 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 4021 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4081 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 4141 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 4201 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg ggctaactac 4261 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 4321 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 4381 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 4441 tctacggggt ctgacgctca gtggaacgac gcgcgcgtaa ctcacgttaa gggattttgg 4501 tcatgagctt gcgccgtccc gtcaagtcag cgtaatgctc tgctttt //