LOCUS       sequence-166352-        4404 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-4 (Chr X: 
            236336..237310).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4404)
  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 4404)
  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: pCfB3035(X-4 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4404
                     /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(1878..2043)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1964..1988
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      2102..2291
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2308..2327)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2309..2327)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2337..2353)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2346..2368)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2902..2935)
                     /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(3405..3421)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3520..3537)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3691..4279)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3771..3790)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4403..4404,1..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 gtttaatccc
     1321 aaagctaaga gtcccatttt attcttctat atgtatattt tcgatactct aaaccaccct
     1381 acaatgtagc cctatactaa atctgctcaa ttttcagctt ctacaagtga ctcgaggcca
     1441 cgtggaaaga tccaactact ccagcacaac gattcaatat aatcgattgc tccactcata
     1501 agaggcaaga acaagcttca acttttggta agccgccgtt tataaacagg gaagatgtcc
     1561 tttgtcaagg gaggcacaga gcatggccaa tttggcaaat tgcaggtttt tctgagtgaa
     1621 aaatgaaaaa gcattgtagt agagtcggct cactgaaaaa ccggggagga cgaaaaggtt
     1681 tccagccaca gttgtagtca cgtgcgcgcc atgctgacta atggcagccg tcgttgggca
     1741 gaagagaatt agtatggtac aggatacgct aattgcgctc caactaccaa ggttgttgag
     1801 ggaacactgg ggcaataggc tgtcgccatt caagagcaga ttaagtcctc agcgagctcg
     1861 catggaatgc gtgcgatgag cgacctcatg ctatacctga gaaagcaacc tgacctacag
     1921 gaaagagtta ctcaagaata agaattttcg ttttaaaacc taagagtcac tttaaaattt
     1981 gtatacactt atttttttta taacttattt aataataaaa atcataaatc ataagaaatt
     2041 cgcttattta gaagtgtcaa caacgtatct accaacggaa tgcgtgcgat cgcgtgcatt
     2101 catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt
     2161 tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc
     2221 tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg
     2281 gacgctcgaa gatcgcgtcc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     2341 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccctgc aggactagtg
     2401 ctgaggcatt aatacatggg aagattcgct tttttttttt gaattacaat agtatgtctg
     2461 atgtctgcaa gaagtaacag gcgtgtgcac aagaatacgt gtgtgtgcgt aagcgtatgc
     2521 actggtggca taacttatct aagaagtata tatcactgac atagaaatgt agatatacag
     2581 gtatttttct cgataatcga taaaaatctc gtcgcgctga accaaacttg gtggttacgg
     2641 agagtttttc tctcatcatt actgtctttc gcattgattt cccctttgac cgataaaatc
     2701 ccttggattc ataagattaa acaaagaggt gatcaaagag aaccctgtga aagtttatgt
     2761 ttataaccgg gcataaagtg aactagacac tttcaagaag ccaaccaaag catgagtaac
     2821 gaagcttacc agcatgatca taccgtaaat cctcaccaga ttaagacctc agcgcggccg
     2881 caaatttaaa taaaatgaag tgaagttcct atactttcta gagaatagga acttctatag
     2941 tgagtcgaat aagggcgaca caaaatttat tctaaatgca taataaatac tgataacatc
     3001 ttatagtttg tattatattt tgtattatcg ttgacatgta taattttgat atcaaaaact
     3061 gattttccct ttattatttt cgagatttat tttcttaatt ctctttaaca aactagaaat
     3121 attgtatata caaaaaatca taaataatag atgaatagtt taattatagg tgttcatcaa
     3181 tcgaaaaagc aacgtatctt atttaaagtg cgttgctttt ttctcattta taaggttaaa
     3241 taattctcat atatcaagca aagtgacagg cgcccttaaa tattctgaca aatgctcttt
     3301 ccctaaactc cccccataaa aaaacccgcc gaagcgggtt tttacgttat ttgcggatta
     3361 acgattactc gttatcagaa ccgcccaggg ggcccgagct taagactggc cgtcgtttta
     3421 caacacagaa agagtttgta gaaacgcaaa aaggccatcc gtcaggggcc ttctgcttag
     3481 tttgatgcct ggcagttccc tactctcgcc ttccgcttcc tcgctcactg actcgctgcg
     3541 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3601 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3661 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3721 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3781 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3841 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3901 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3961 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4021 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4081 cggtgctaca gagttcttga agtggtgggc taactacggc tacactagaa gaacagtatt
     4141 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4201 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4261 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4321 gaacgacgcg cgcgtaactc acgttaaggg attttggtca tgagcttgcg ccgtcccgtc
     4381 aagtcagcgt aatgctctgc tttt
//
