LOCUS       sequence-134828-        4567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone-MarkerFree backbone vector for the addition of 1 or 2 
            genes and promoters for integration into site XII-5 (Chr XII: 
            839226..840357).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4567)
  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 4567)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1002/biot.201600147"; journalName: "Biotechnol J"; date: 
            "2016-08"; volume: "11"; issue: "8"; pages: "1110-7"
COMMENT     Sequence Label: pCfB2909(XII-5 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..30)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(12..30)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(40..56)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(49..71)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(835..868)
                     /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(1338..1354)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1453..1470)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1624..2212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1704..1723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2336..3127,3128..3196))
                     /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     2959..2978
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3197..3288)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3330..3349
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3355..3374
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    3581..3614
                     /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      complement(4147..4312)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     4233..4257
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      4371..4560
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
ORIGIN
        1 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
       61 tcgtgactgg gaaaaccctg gcgttacccc tgcaggacta gtgctgaggc attaatgcaa
      121 ctcagaagtt tgacagcaag caagttcatc attcgaacta gccttattgt tttagttcag
      181 tgacagcgaa ctgccgtact cgatgcttta tttctcacgg tagagcggaa gaacagatag
      241 gggcagcgtg agaagagtta gaaagtaaat ttttatcacg tctgaagtat tcttattcat
      301 aggaaatttt gcaaggtttt ttagctcaat aacgggctaa gttatataag gtgttcacgc
      361 gattttcttg ttatgtatac ctcttctctg aggaatggta ctactgtcct gatgtaggct
      421 ccttaaattg gtgggcaaga ataacttatc gatattttgt atattggtct tggagttcac
      481 cacgtaatgc ctgtttaaga ccatcagtta actctagtat tatttggtct tggctactgg
      541 ccgtttgcta ttattcaagt cttttgtgcc ttcccgtcgg gtaagggagt tatttaggga
      601 tacagaatct aacgaaaact aaatctcaat gattaactct atttaatcct tttttgaaag
      661 gcaaaagagg tcccttgttc acttacaacg ttcttagcca aattcgctta tcacttacta
      721 cttcacgata tacagaagta aaaacatata aaaagatgtc tgtttgttta gccatcacaa
      781 aaggtatcgc agattaagac ctcagcgcgg ccgcaaattt aaataaaatg aagtgaagtt
      841 cctatacttt ctagagaata ggaacttcta tagtgagtcg aataagggcg acacaaaatt
      901 tattctaaat gcataataaa tactgataac atcttatagt ttgtattata ttttgtatta
      961 tcgttgacat gtataatttt gatatcaaaa actgattttc cctttattat tttcgagatt
     1021 tattttctta attctcttta acaaactaga aatattgtat atacaaaaaa tcataaataa
     1081 tagatgaata gtttaattat aggtgttcat caatcgaaaa agcaacgtat cttatttaaa
     1141 gtgcgttgct tttttctcat ttataaggtt aaataattct catatatcaa gcaaagtgac
     1201 aggcgccctt aaatattctg acaaatgctc tttccctaaa ctccccccat aaaaaaaccc
     1261 gccgaagcgg gtttttacgt tatttgcgga ttaacgatta ctcgttatca gaaccgccca
     1321 gggggcccga gcttaagact ggccgtcgtt ttacaacaca gaaagagttt gtagaaacgc
     1381 aaaaaggcca tccgtcaggg gccttctgct tagtttgatg cctggcagtt ccctactctc
     1441 gccttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     1501 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     1561 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     1621 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     1681 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     1741 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     1801 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     1861 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     1921 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     1981 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2041 ggctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     2101 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2161 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2221 tttgatcttt tctacggggt ctgacgctca gtggaacgac gcgcgcgtaa ctcacgttaa
     2281 gggattttgg tcatgagctt gcgccgtccc gtcaagtcag cgtaatgctc tgcttttacc
     2341 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2401 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagcg
     2461 ctgcgatgat accgcgagaa ccacgctcac cggctccgga tttatcagca ataaaccagc
     2521 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2581 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2641 ttgccatcgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2701 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2761 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2821 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2881 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2941 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3001 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3061 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3121 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3181 aatgttgaat actcatattc ttcctttttc aatattattg aagcatttat cagggttatt
     3241 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggtcagtg
     3301 ttacaaccaa ttaaccaatt ctgaacatta tcgcgagccc atttatacct gaatatggct
     3361 cataacaccc cttgtttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     3421 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggactcccca tgcgagagta
     3481 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgccc
     3541 gggctaatta tggggtgtcg cccttattcg actctatagt gaagttccta ttctctagaa
     3601 agtataggaa cttctgaagt ggggatttaa atgcggccgc gctgagggtt taatcaatct
     3661 ggcggcttga gttctcaaca tgttttattt tttacttata ttgctggtag ggtaaaaaaa
     3721 tataactcct aggaataggt tgtctatatg tttttgtctt gcttctataa ttgtaacaaa
     3781 caaggaaagg gaaaatactg ggtgtaaaag ccattgagtc aagttaggtc atccctttta
     3841 tacaaaattt ttcaattttt tttccaagat tcttgtacga ttaattattt tttttttgcg
     3901 tcctacagcg tgatgaaaat ttcgcctgct gcaagatgag cgggaacggg cgaaatgtgc
     3961 acgcgcacaa cttacgaaac gcggatgagt cactgacagc caccgcagag gttctgactc
     4021 ctactgagct ctattggagg tggcagaacc ggtaccggag gaggccgcta taaccggttt
     4081 gaatttattg tcacagtgtc acatcagcat taagtcctca gcgagctcgc atggaatgcg
     4141 tgcgatgagc gacctcatgc tatacctgag aaagcaacct gacctacagg aaagagttac
     4201 tcaagaataa gaattttcgt tttaaaacct aagagtcact ttaaaatttg tatacactta
     4261 ttttttttat aacttattta ataataaaaa tcataaatca taagaaattc gcttatttag
     4321 aagtgtcaac aacgtatcta ccaacggaat gcgtgcgatc gcgtgcattc atccgctcta
     4381 accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt
     4441 atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc
     4501 gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag
     4561 atcgcgt
//
