LOCUS       sequence-166358-        4539 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-1 (Chr XII: 
            795787..796720).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4539)
  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 4539)
  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: pCfB3038(XII-1 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4539
                     /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(1870..2035)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1956..1980
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      2094..2283
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2300..2319)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2301..2319)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2329..2345)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2338..2360)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(3037..3070)
                     /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(3540..3556)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3655..3672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3826..4414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3906..3925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4538..4539,1..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 gtttaatgtt
     1321 gagctctgtc cttcatggac tttttggacc acttcttctt ggacttctta ccaccagcaa
     1381 gagcggcagc ggctttggca gctttagaca attgttgctt tggaggcatg ttatattatg
     1441 ttctgagacg taagaaaggg tgaaaattga tgttagtgtc aaaaattata ttacaaaata
     1501 cgcagagata ttctagttcc tttgatgaat gaatctttca gaaaaaaaag tcaaagcaaa
     1561 agcaaaatgg cctgcagact aaactgtatg gtggtcttgg aatgataaag atctgtttaa
     1621 tagatttagt agatacaata gcacatctca ttacccagtt atgattgacg tcattctgag
     1681 ttacaatgat cttagcgacc tgtgctcatt atttgctcca ctaattctaa ttttcctcgc
     1741 ctttcatatt tcgtatcttt attctatatc ctaaaatttt tttggcaaat cccagatttg
     1801 gctttgattt tggcatcggt tcggttcttt cattaagtcc tcagcgagct cgcatggaat
     1861 gcgtgcgatg agcgacctca tgctatacct gagaaagcaa cctgacctac aggaaagagt
     1921 tactcaagaa taagaatttt cgttttaaaa cctaagagtc actttaaaat ttgtatacac
     1981 ttattttttt tataacttat ttaataataa aaatcataaa tcataagaaa ttcgcttatt
     2041 tagaagtgtc aacaacgtat ctaccaacgg aatgcgtgcg atcgcgtgca ttcatccgct
     2101 ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata
     2161 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga
     2221 cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg
     2281 aagatcgcgt cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     2341 tttacaacgt cgtgactggg aaaaccctgg cgttacccct gcaggactag tgctgaggca
     2401 ttaatcttcc cgtgaatcaa ctgcacatac cttaataact agttcagttt agtgctctgt
     2461 ctgagtgact gtaataataa attttacaag ccacttctca tgacatatat tggtaagtaa
     2521 cttcatcaat actaattagt ctttgccggt tacccatctg gcccctgact tgcgatgctt
     2581 aggaagttcc atactcgcgg ctcttcccaa cagtagcaca tccgtgaaac ttctggcgct
     2641 attcattatg cagtaccagg acaagaagtt aaaaaaaaag ctctgttaca agttcaatgg
     2701 tggtgcaagg attgaagtta ttatccagga ggcacggatg caaaagatag aggacaaatt
     2761 aatttcctta aaattcaaaa tgctcattat tgtcacgctg tatgagcatt tggtgaagat
     2821 ttcactggga aatgttgcaa taatttgata atcgttcgta ttggatgaaa ctgtaacatc
     2881 atctgtttat taagtatccg tgttattagt atatcatcac atacggtgta agaagataac
     2941 ataaagattg agaaacagtc atcaaatata atggaagctg aaatgcgagg attgattaag
     3001 acctcagcgc ggccgcaaat ttaaataaaa tgaagtgaag ttcctatact ttctagagaa
     3061 taggaacttc tatagtgagt cgaataaggg cgacacaaaa tttattctaa atgcataata
     3121 aatactgata acatcttata gtttgtatta tattttgtat tatcgttgac atgtataatt
     3181 ttgatatcaa aaactgattt tccctttatt attttcgaga tttattttct taattctctt
     3241 taacaaacta gaaatattgt atatacaaaa aatcataaat aatagatgaa tagtttaatt
     3301 ataggtgttc atcaatcgaa aaagcaacgt atcttattta aagtgcgttg cttttttctc
     3361 atttataagg ttaaataatt ctcatatatc aagcaaagtg acaggcgccc ttaaatattc
     3421 tgacaaatgc tctttcccta aactcccccc ataaaaaaac ccgccgaagc gggtttttac
     3481 gttatttgcg gattaacgat tactcgttat cagaaccgcc cagggggccc gagcttaaga
     3541 ctggccgtcg ttttacaaca cagaaagagt ttgtagaaac gcaaaaaggc catccgtcag
     3601 gggccttctg cttagtttga tgcctggcag ttccctactc tcgccttccg cttcctcgct
     3661 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3721 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3781 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3841 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3901 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3961 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4021 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4081 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4141 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4201 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tgggctaact acggctacac
     4261 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4321 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4381 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4441 gtctgacgct cagtggaacg acgcgcgcgt aactcacgtt aagggatttt ggtcatgagc
     4501 ttgcgccgtc ccgtcaagtc agcgtaatgc tctgctttt
//
