LOCUS       sequence-166349-        4549 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-3 (Chr X: 
            223616..224744).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4549)
  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 4549)
  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: pCfB3034(X-3 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4549
                     /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(1928..2093)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2014..2038
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      2152..2341
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2358..2377)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2359..2377)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2387..2403)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2396..2418)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(3047..3080)
                     /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(3550..3566)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3665..3682)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3836..4424)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3916..3935)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4548..4549,1..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 gtttaatcga
     1321 gatctttgtg ttcggttacc cggctcagat cctaacttct tcttttggta tgtttattcg
     1381 tataagttac tgttgtccac aggcaatact ctgcagaaaa ttaaaacggc attaatgcta
     1441 ggacaaccag aattgttact actgtatgtg cgatagttga taactgcaac attatgcccg
     1501 gtatattctc aaaaaaccct attactgcat acgaagaaat cgcaagagaa atctttcggt
     1561 ttggaaaagc tcactgtgag gttccttgga gccaatagta atacagcaca atccaaggaa
     1621 aaatctggcc tatatgcaag gaaggagaga tagtcaaaag cattctttcc cctagaagtt
     1681 ggtgcatata tggcatcgtt aaaacatatt acccccaaaa tttcttctct aaacgatgtg
     1741 cttggccttt gttttggttt ttgatgtcgg tcgtttgagg ccccttgcgg aaaatcgaga
     1801 tcgccgaatg gcacgcgagg gaagggaaat aaggtttaaa ggcactgaaa caataggcaa
     1861 gaagtaggcg agagccgaca tacgagacta ttaagtcctc agcgagctcg catggaatgc
     1921 gtgcgatgag cgacctcatg ctatacctga gaaagcaacc tgacctacag gaaagagtta
     1981 ctcaagaata agaattttcg ttttaaaacc taagagtcac tttaaaattt gtatacactt
     2041 atttttttta taacttattt aataataaaa atcataaatc ataagaaatt cgcttattta
     2101 gaagtgtcaa caacgtatct accaacggaa tgcgtgcgat cgcgtgcatt catccgctct
     2161 aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt
     2221 tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg
     2281 cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa
     2341 gatcgcgacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     2401 tacaacgtcg tgactgggaa aaccctggcg ttacccctgc aggactagtg ctgaggcatt
     2461 aatccacttt tcaatgaaac ggatattgat atgctagtaa aaggacgagc tcaagagcga
     2521 aaatataagt aaagaattcg agtgcacttg tctccatgca gcaagatttc atatgagtct
     2581 tttttatctt tttacttttt acattacacg atatgcactt tatgaaaatt taacgaggtt
     2641 ggaagccgga taatcaacca aaatcaggca cgaaggcaca ctcgtatatg catgttgttg
     2701 aaactctgtt acgctgaact aacaatcaca catgtagagg tcaccgggaa aagttgcgac
     2761 cccatggaag gtcgatctct tcgtttggct ttgcttggct ggcggcattg cgcttcttcg
     2821 cttatacccg tctcttgacg ctcgagctcg ttcattgaga tacctttatt cttgcacatt
     2881 ttctggcttt tttcgctact cgggtacatg taatcatgca cacagaaggt gctgtagggt
     2941 gaaagttcct ttgtgctgtc gtttgttttt aatgccaaac tttccggtga tcaataacca
     3001 cctcattaag acctcagcgc ggccgcaaat ttaaataaaa tgaagtgaag ttcctatact
     3061 ttctagagaa taggaacttc tatagtgagt cgaataaggg cgacacaaaa tttattctaa
     3121 atgcataata aatactgata acatcttata gtttgtatta tattttgtat tatcgttgac
     3181 atgtataatt ttgatatcaa aaactgattt tccctttatt attttcgaga tttattttct
     3241 taattctctt taacaaacta gaaatattgt atatacaaaa aatcataaat aatagatgaa
     3301 tagtttaatt ataggtgttc atcaatcgaa aaagcaacgt atcttattta aagtgcgttg
     3361 cttttttctc atttataagg ttaaataatt ctcatatatc aagcaaagtg acaggcgccc
     3421 ttaaatattc tgacaaatgc tctttcccta aactcccccc ataaaaaaac ccgccgaagc
     3481 gggtttttac gttatttgcg gattaacgat tactcgttat cagaaccgcc cagggggccc
     3541 gagcttaaga ctggccgtcg ttttacaaca cagaaagagt ttgtagaaac gcaaaaaggc
     3601 catccgtcag gggccttctg cttagtttga tgcctggcag ttccctactc tcgccttccg
     3661 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3721 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3781 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3841 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3901 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3961 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4021 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4081 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4141 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4201 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tgggctaact
     4261 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4321 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4381 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4441 tttctacggg gtctgacgct cagtggaacg acgcgcgcgt aactcacgtt aagggatttt
     4501 ggtcatgagc ttgcgccgtc ccgtcaagtc agcgtaatgc tctgctttt
//
