LOCUS       sequence-166065-        4520 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-2 (Chr XII: 
            808805..809939).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4520)
  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 4520)
  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: pCfB3039(XII-2 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(64..855,856..924))
                     /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     687..706
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(925..1016)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1058..1077
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1083..1102
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1309..1342
                     /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(1956..2121)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2042..2066
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      2180..2369
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2386..2405)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2387..2405)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2415..2431)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2424..2446)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(3083..3116)
                     /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(3586..3602)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3701..3718)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3872..4460)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3952..3971)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cacgttaagg gattttggtc atgagcttgc gccgtcccgt caagtcagcg taatgctctg
       61 cttttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
      121 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
      181 ccccagcgct gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat
      241 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
      301 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
      361 caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
      421 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
      481 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
      541 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
      601 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
      661 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
      721 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
      781 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
      841 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
      901 gacacggaaa tgttgaatac tcatattctt cctttttcaa tattattgaa gcatttatca
      961 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     1021 ggtcagtgtt acaaccaatt aaccaattct gaacattatc gcgagcccat ttatacctga
     1081 atatggctca taacacccct tgtttgcctg gcggcagtag cgcggtggtc ccacctgacc
     1141 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg actccccatg
     1201 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     1261 tttcgcccgg gctaattatg gggtgtcgcc cttattcgac tctatagtga agttcctatt
     1321 ctctagaaag tataggaact tctgaagtgg ggatttaaat gcggccgcgc tgagggttta
     1381 atcggcatgc aaacatctac acaattagca agggcaatcc atattttgtc ttttcgcgcc
     1441 ctggaaaggc ctaagtaatg tcgtaaacgc attctatctg tacttcaact ctcctctgtg
     1501 cattggtttg tgcaaatcac attttacgat actgccagat atatgcaaaa agagaaaacc
     1561 aagggaccag aacaaagcaa aattacgata ttcttcgaat tccttcgtgc ttgactaaga
     1621 caaagggatg gacgtagcga tttttagcgg gccaagaact ggttccgaaa aagcacaggt
     1681 acaccgaacc ctcagctaag gagggacagc accgatgcgg aaggacaaac tttctttttg
     1741 cctatcacag tatcttatcg agctaactat tttcgacaca catgaaaaag cagaaatatt
     1801 aacgaaaaag aaaagaaaga ccatgtcatg tacgggcaat cagaatctgt aacaagcgcc
     1861 attttttttt ctgtatcggg ccctccttac tgctctcctt ccgtgtaacg cgttatgatt
     1921 aagtcctcag cgagctcgca tggaatgcgt gcgatgagcg acctcatgct atacctgaga
     1981 aagcaacctg acctacagga aagagttact caagaataag aattttcgtt ttaaaaccta
     2041 agagtcactt taaaatttgt atacacttat tttttttata acttatttaa taataaaaat
     2101 cataaatcat aagaaattcg cttatttaga agtgtcaaca acgtatctac caacggaatg
     2161 cgtgcgatcg cgtgcattca tccgctctaa ccgaaaagga aggagttaga caacctgaag
     2221 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc
     2281 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct
     2341 tgcttgagaa ggttttggga cgctcgaaga tcgcgtccca attcgcccta tagtgagtcg
     2401 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     2461 acccctgcag gactagtgct gaggcattaa tcgactctct cgaaattttt cttaacgcgt
     2521 ccttgtactg cgtctaacgc ttttgccact tggatttcta ttataggaaa tagtctcact
     2581 tactgggcga cgaattttcg cgttttgatg aagcacagga agaatttctt ttttttttgg
     2641 cttcttctgg ttccgttttt tacgcgcaca aatctaaaaa aagaaataat tataacctag
     2701 tctcgaaaat tttcatcgat ccattcgttc ctttttttcg attttttcag atcaaaattc
     2761 ttgtttcttt ctttgtctta gtttatatta aaagatattt tgattttact cctgaactat
     2821 ttattctttc taagaaggcc agaacactac agctgtttta accgactacg aagttctcca
     2881 ttctcgaaca ctagccttca tttaccaaac aggaactagc gtatatcatt agtccttatt
     2941 cgaaaagaga ttggtagata tttattgtag tttgtgagaa ggagaaaata ctgtcattgg
     3001 actgatagtt agaggacatt aacctctctt acgttcgctc attaagacct cagcgcggcc
     3061 gcaaatttaa ataaaatgaa gtgaagttcc tatactttct agagaatagg aacttctata
     3121 gtgagtcgaa taagggcgac acaaaattta ttctaaatgc ataataaata ctgataacat
     3181 cttatagttt gtattatatt ttgtattatc gttgacatgt ataattttga tatcaaaaac
     3241 tgattttccc tttattattt tcgagattta ttttcttaat tctctttaac aaactagaaa
     3301 tattgtatat acaaaaaatc ataaataata gatgaatagt ttaattatag gtgttcatca
     3361 atcgaaaaag caacgtatct tatttaaagt gcgttgcttt tttctcattt ataaggttaa
     3421 ataattctca tatatcaagc aaagtgacag gcgcccttaa atattctgac aaatgctctt
     3481 tccctaaact ccccccataa aaaaacccgc cgaagcgggt ttttacgtta tttgcggatt
     3541 aacgattact cgttatcaga accgcccagg gggcccgagc ttaagactgg ccgtcgtttt
     3601 acaacacaga aagagtttgt agaaacgcaa aaaggccatc cgtcaggggc cttctgctta
     3661 gtttgatgcc tggcagttcc ctactctcgc cttccgcttc ctcgctcact gactcgctgc
     3721 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3781 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3841 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3901 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3961 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4021 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4081 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4141 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4201 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4261 gcggtgctac agagttcttg aagtggtggg ctaactacgg ctacactaga agaacagtat
     4321 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4381 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4441 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4501 ggaacgacgc gcgcgtaact
//
