LOCUS       sequence-166354-        4547 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 XI-3 (Chr XI: 
            93378..94567).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4547)
  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 4547)
  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: pCfB2904(XI-3 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4547
                     /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(1931..2096)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2017..2041
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      2155..2344
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2361..2380)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2362..2380)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2390..2406)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2399..2421)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(3045..3078)
                     /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(3548..3564)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3663..3680)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3834..4422)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3914..3933)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4546..4547,1..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 gtttaatagt
     1321 tacttgctct atgcgtttgc gcatcctctt tttacttttt ttttttcagt aaagcctaag
     1381 cataaatcgt tttatacgta cgacacgttc aacttttctt ggttagtagt ggcaatctct
     1441 gcaatacata cagggagtca tggtctatca tcttgtccaa tcaaagaagc atcggttcag
     1501 atcgagcaaa ctgtagggag aaaggaaagt agaaatgcag agtgtgctat atgtccaatc
     1561 tcggttttgt agtttggatg tcattagaga tctaccaccc aaccggctgc tttcatgtgg
     1621 aacagaaaag aaatcggggc gcttcctctt ctgtattcct ttagttaacg tttttattca
     1681 gccatctaac catcataccc ccatacggta acaaaacctc ttctaagaaa agaagtctct
     1741 gctcctccgc catcttattt ttattcgctg cgcgcgttta ttgtcgcatc gctggccagc
     1801 aaaaaattgg ttgccttttt ttacctaaaa aagacacatc taactgatta gttttccgtt
     1861 ttaggatatt gacgccaagc gtgcgtctga ttattaagtc ctcagcgagc tcgcatggaa
     1921 tgcgtgcgat gagcgacctc atgctatacc tgagaaagca acctgaccta caggaaagag
     1981 ttactcaaga ataagaattt tcgttttaaa acctaagagt cactttaaaa tttgtataca
     2041 cttatttttt ttataactta tttaataata aaaatcataa atcataagaa attcgcttat
     2101 ttagaagtgt caacaacgta tctaccaacg gaatgcgtgc gatcgcgtgc attcatccgc
     2161 tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat
     2221 agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag
     2281 acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc
     2341 gaagatcgcg tcccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     2401 ttttacaacg tcgtgactgg gaaaaccctg gcgttacccc tgcaggacta gtgctgaggc
     2461 attaatttac gtggattgag ccagcaatac agatcattat taaattgttt tgtacatgat
     2521 gttagtatat aatcgtaaag cttttctaat atgtatacct tatacatgga actccacaga
     2581 acttgcaaac ataccaaaaa tcccttattc ttgttcactc attttacatc aaaaaataat
     2641 atttcagtta ttaaggaaaa taaaaaaata gattagagaa gcattttgaa gaaatagtat
     2701 attcttttat tgaacctaag agcgtgatat ttttactcga aataaaatac gaaaaatcta
     2761 tgcactcatc tttccgacta ctattggctc ctgctcaaaa aaagagggaa aaaaagctcc
     2821 aaaattctat cttttcctat cgctcctgtc ctatccttat tacgttcatt actattttaa
     2881 tactatccat tcttttattt tcagtctaaa aaaaacattt ctcataacgg gaaaagcaaa
     2941 aaaatgtcaa gcttatacat caaaacacca ctgcatgcat tatctgctgg tccggattct
     3001 caattaagac ctcagcgcgg ccgcaaattt aaataaaatg aagtgaagtt cctatacttt
     3061 ctagagaata ggaacttcta tagtgagtcg aataagggcg acacaaaatt tattctaaat
     3121 gcataataaa tactgataac atcttatagt ttgtattata ttttgtatta tcgttgacat
     3181 gtataatttt gatatcaaaa actgattttc cctttattat tttcgagatt tattttctta
     3241 attctcttta acaaactaga aatattgtat atacaaaaaa tcataaataa tagatgaata
     3301 gtttaattat aggtgttcat caatcgaaaa agcaacgtat cttatttaaa gtgcgttgct
     3361 tttttctcat ttataaggtt aaataattct catatatcaa gcaaagtgac aggcgccctt
     3421 aaatattctg acaaatgctc tttccctaaa ctccccccat aaaaaaaccc gccgaagcgg
     3481 gtttttacgt tatttgcgga ttaacgatta ctcgttatca gaaccgccca gggggcccga
     3541 gcttaagact ggccgtcgtt ttacaacaca gaaagagttt gtagaaacgc aaaaaggcca
     3601 tccgtcaggg gccttctgct tagtttgatg cctggcagtt ccctactctc gccttccgct
     3661 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3721 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3781 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3841 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3901 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3961 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4021 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4081 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4141 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4201 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg ggctaactac
     4261 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4321 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4381 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4441 tctacggggt ctgacgctca gtggaacgac gcgcgcgtaa ctcacgttaa gggattttgg
     4501 tcatgagctt gcgccgtccc gtcaagtcag cgtaatgctc tgctttt
//
