LOCUS       sequence-166353-        4474 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-1 (Chr XI: 
            67491..68573).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4474)
  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 4474)
  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: pCfB3036(XI-1 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4474
                     /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(1817..1982)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1903..1927
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      2041..2230
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2248..2267)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2249..2267)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2277..2293)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2286..2308)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2972..3005)
                     /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(3475..3491)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3590..3607)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3761..4349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3841..3860)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4473..4474,1..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 gtttaatgga
     1321 atagtgacgt tgtgatgcgg tgagttcggc ggttagggga atggtatatg ataaaaaacg
     1381 gaaacgtgct tctttaattt aattgtttaa tattgttgca gatatataaa aagggggaaa
     1441 gaaccgaaga tgtaattatt tttttatcgc ctcaacctaa agcaagcaat aaggtataaa
     1501 gatcaggacg tctcgagcgc tgatatctaa atttgaagcc acgcaagtaa ctacgtaggt
     1561 cagaggggac aaggaataac acttgacatt tttctttttt cttttttttt cttttttttt
     1621 tttttgttaa tcttggcttc tgtaccgtag cctcctgtat acggttttag atgcagtatg
     1681 tttatcatcg ccgagaaatt tggggggtgc aaaggaataa aatccgtgct gaaatccgtg
     1741 caccgcatca aactttctcg gaggattctt tgcagccgat taagtcctca gcgagctcgc
     1801 atggaatgcg tgcgatgagc gacctcatgc tatacctgag aaagcaacct gacctacagg
     1861 aaagagttac tcaagaataa gaattttcgt tttaaaacct aagagtcact ttaaaatttg
     1921 tatacactta ttttttttat aacttattta ataataaaaa tcataaatca taagaaattc
     1981 gcttatttag aagtgtcaac aacgtatcta ccaacggaat gcgtgcgatc gcgtgcattc
     2041 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt
     2101 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct
     2161 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
     2221 acgctcgaag atcgcgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     2281 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccctgc aggactagtg
     2341 ctgaggcatt aatagtttct tggcattggc aaatctctgc taaatgctgc gtacagacgg
     2401 aaactcacac cgccgcgaaa actggtcagt ggcaaaaaaa aaaaaaatta aaaaaataaa
     2461 aaataactat tacgtatgat actgtttctg gtagttgata tgaggttggt gttgtatatt
     2521 gtacgtttta ggaacaggga agtgaatatt atttactctg ctgcacattc tggctaggtc
     2581 gaagccggaa cctgagatga cgccgcgcta gaactatgga ccaagctgtt gacaatgttt
     2641 agatggtgat gcactaccct gtgcggggag tggccacgaa cgcgagcgga aggtgcgggt
     2701 gttgcgggaa ttgcgggagg ttcttcgcta agcgtgaggg ttgctagttg gggcggcggg
     2761 gtttccctaa gtgtaaatag ggcctcgccg ctggcacatg agtgcgccgg aggaggcggc
     2821 ggaggcgacg acgctaaaac cgtggccgtt ggggaaggat gggcggctat atctaccatt
     2881 gacctgatgg ggactcggct cttaaggaat gggtttgagg tgggtgtgga ttaagacctc
     2941 agcgcggccg caaatttaaa taaaatgaag tgaagttcct atactttcta gagaatagga
     3001 acttctatag tgagtcgaat aagggcgaca caaaatttat tctaaatgca taataaatac
     3061 tgataacatc ttatagtttg tattatattt tgtattatcg ttgacatgta taattttgat
     3121 atcaaaaact gattttccct ttattatttt cgagatttat tttcttaatt ctctttaaca
     3181 aactagaaat attgtatata caaaaaatca taaataatag atgaatagtt taattatagg
     3241 tgttcatcaa tcgaaaaagc aacgtatctt atttaaagtg cgttgctttt ttctcattta
     3301 taaggttaaa taattctcat atatcaagca aagtgacagg cgcccttaaa tattctgaca
     3361 aatgctcttt ccctaaactc cccccataaa aaaacccgcc gaagcgggtt tttacgttat
     3421 ttgcggatta acgattactc gttatcagaa ccgcccaggg ggcccgagct taagactggc
     3481 cgtcgtttta caacacagaa agagtttgta gaaacgcaaa aaggccatcc gtcaggggcc
     3541 ttctgcttag tttgatgcct ggcagttccc tactctcgcc ttccgcttcc tcgctcactg
     3601 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3661 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3721 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3781 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3841 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3901 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3961 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4021 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4081 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4141 ggtatgtagg cggtgctaca gagttcttga agtggtgggc taactacggc tacactagaa
     4201 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4261 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4321 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4381 acgctcagtg gaacgacgcg cgcgtaactc acgttaaggg attttggtca tgagcttgcg
     4441 ccgtcccgtc aagtcagcgt aatgctctgc tttt
//
