LOCUS       sequence-165975-        4457 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 X-2 (Chr X: 
            194944..195980).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4457)
  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 4457)
  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: pCfB2899(X-2 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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      complement(1887..2052)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1973..1997
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      2111..2300
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2317..2336)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2318..2336)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2346..2362)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2355..2377)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2955..2988)
                     /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(3458..3474)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3573..3590)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3744..4332)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3824..3843)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4456..4457,1..790,791..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 gtttaatcgt
     1321 ctatgaggag actgttagtt ggatatcagt aatgagacga aaaagctcga aatgaatgga
     1381 tatattcttt ttgctactgg caactgttga atatttaatg ttaaaacaaa ctaactgagg
     1441 tatattcgta tctgtatgta cacatatact atatacagga aaagataagc aagagagagg
     1501 atatcaacta cgagagcgat cgattatata tcaaaagctg tccgctttgc cacccataat
     1561 cggcgcttag tttcggagtt caatcataat tctaccacct tacactcaac ttactcttta
     1621 actcctatag tataatatcg ccactgaccc catattaaaa aatttttttg ctcgatcttc
     1681 tatcctcttt aggttaattg tcgctgttat tgtctagatt ttttctcgga gatggcgcat
     1741 ctatttgccg tcaaaagatc ctctcatacc atattaagta aattgcctcc atttcttttt
     1801 cctcgggcag agaaactcgc aggcaacttg ctctcgaagt ggtcacgtat taagtcctca
     1861 gcgagctcgc atggaatgcg tgcgatgagc gacctcatgc tatacctgag aaagcaacct
     1921 gacctacagg aaagagttac tcaagaataa gaattttcgt tttaaaacct aagagtcact
     1981 ttaaaatttg tatacactta ttttttttat aacttattta ataataaaaa tcataaatca
     2041 taagaaattc gcttatttag aagtgtcaac aacgtatcta ccaacggaat gcgtgcgatc
     2101 gcgtgcattc atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc
     2161 ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc
     2221 ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga
     2281 aggttttggg acgctcgaag atcgcgtccc aattcgccct atagtgagtc gtattacgcg
     2341 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccctgca
     2401 ggactagtgc tgaggcatta atcctgcata atcggcctca cagagggatc ccgttaccca
     2461 tctatgctga agatttatca tactattcct ccgctcgttt cttttttcag tgaggtgtgt
     2521 cgtgaaagaa aacccacaat taaactttca acaaccgggc gactaggaag agagtagtgg
     2581 gcgcggatga cgaaggctaa ggtcacttct tcgtttcctt tattggggtt tccgtgtagc
     2641 cttcccctga atagtgtggg acgttttatg agaagccgta agaaataggc aaattgagtt
     2701 atgacaagta gacatgatgc cgcagccttg cctgacttta cgtctccttc atgaataagt
     2761 ttttctatcg agttcttttc cttttttcgc cttaattagc tcaattaagc ctgtcctcac
     2821 tacttttctt tttcttatcg gctttgtgcc acacctaacc ttcgaatgct gttttattcc
     2881 gttcttacat gggatggtaa tgccttggcg agattaagac ctcagcgcgg ccgcaaattt
     2941 aaataaaatg aagtgaagtt cctatacttt ctagagaata ggaacttcta tagtgagtcg
     3001 aataagggcg acacaaaatt tattctaaat gcataataaa tactgataac atcttatagt
     3061 ttgtattata ttttgtatta tcgttgacat gtataatttt gatatcaaaa actgattttc
     3121 cctttattat tttcgagatt tattttctta attctcttta acaaactaga aatattgtat
     3181 atacaaaaaa tcataaataa tagatgaata gtttaattat aggtgttcat caatcgaaaa
     3241 agcaacgtat cttatttaaa gtgcgttgct tttttctcat ttataaggtt aaataattct
     3301 catatatcaa gcaaagtgac aggcgccctt aaatattctg acaaatgctc tttccctaaa
     3361 ctccccccat aaaaaaaccc gccgaagcgg gtttttacgt tatttgcgga ttaacgatta
     3421 ctcgttatca gaaccgccca gggggcccga gcttaagact ggccgtcgtt ttacaacaca
     3481 gaaagagttt gtagaaacgc aaaaaggcca tccgtcaggg gccttctgct tagtttgatg
     3541 cctggcagtt ccctactctc gccttccgct tcctcgctca ctgactcgct gcgctcggtc
     3601 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3661 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3721 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3781 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3841 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3901 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3961 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4021 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4081 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4141 acagagttct tgaagtggtg ggctaactac ggctacacta gaagaacagt atttggtatc
     4201 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4261 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4321 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgac
     4381 gcgcgcgtaa ctcacgttaa gggattttgg tcatgagctt gcgccgtccc gtcaagtcag
     4441 cgtaatgctc tgctttt
//
