LOCUS       sequence-165988-        4568 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-2 (Chr XI: 
            91575..92913).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4568)
  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 4568)
  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: pCfB2903(XI-2 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4568
                     /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(1925..2090)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2011..2035
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      2149..2338
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2355..2374)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2356..2374)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2384..2400)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2393..2415)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(3066..3099)
                     /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(3569..3585)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3684..3701)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3855..4443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3935..3954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4567..4568,1..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 gtttaatgag
     1321 gattttcgat ggagcaggat gaggagaaat agtaccacat gtatatatcc attacaaaaa
     1381 ggtttatata caattacaat agacccttgt tggggtttct gaaaaaagaa gtagtcgatg
     1441 ccatcggcaa taatacggaa ttacgagaaa cacaatcccg atcctttttt gggtaattac
     1501 ttcaccgatt ctaccgattt atcatgccaa aaaaaattca ccgtgggttc tagaagtgcc
     1561 ctttgaggat tgtagccact ctaacccaca cggcctcctt actagctgac taaggtgaca
     1621 aaaccgcaag gactggaaag tcgccactca tctgaaaatt ctcaagtttt tcactactga
     1681 gtttatgctt tcgaattttt ttgttcggta atagcacggc ggttcgattc aattccgccg
     1741 ctccgagcga tgctccgcaa aactcagtaa taagctttct gatggttcac ccctttttta
     1801 gcacgcgggg tgtaactcaa cagaaaaatg tgccatagaa caagactagg caaaagccaa
     1861 ggagcgtttg ccatgaactt ccacaaatta agtcctcagc gagctcgcat ggaatgcgtg
     1921 cgatgagcga cctcatgcta tacctgagaa agcaacctga cctacaggaa agagttactc
     1981 aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta tacacttatt
     2041 ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc ttatttagaa
     2101 gtgtcaacaa cgtatctacc aacggaatgc gtgcgatcgc gtgcattcat ccgctctaac
     2161 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat
     2221 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt
     2281 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaagat
     2341 cgcgacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     2401 aacgtcgtga ctgggaaaac cctggcgtta cccctgcagg actagtgctg aggcattaat
     2461 cacacgacta gcgctttcag atattaaaaa gtttagatgt aggttttagc ggtaacagtt
     2521 atataaatcg tgtttcttct cttgatgaaa caaaaaaatg ctagaaaaac tttgtcgttt
     2581 cttacttttg gtgcgctttg cagttttcgt ggctagactt agaatcattt ctcctcagat
     2641 ttcttgatta aagtttggtg cgaagcccta ctctaacatt ggtgttcttc ttttcattca
     2701 cgcaagttaa gtccaggaag gtgagcaaat gctcatcctt ctgttcatgc gtgacggctg
     2761 aattatcctt atctggcgta cccgtgcagc cgtttccgtg cctcggttcc tccgagatat
     2821 ccttagggac cgccagggac catgattgcg tcaactgttg tcaccgctcc agaggatcct
     2881 ctgtaacctt ttcaaccata aaaatagaaa gggcacaggg ttggggtatt tgaatttttt
     2941 ttttgggttt tttcgtatta cttattactt accttctttt ctatataatt ttgtttttcc
     3001 ctggtagagc ggaatcttcc cacattaaga cctcagcgcg gccgcaaatt taaataaaat
     3061 gaagtgaagt tcctatactt tctagagaat aggaacttct atagtgagtc gaataagggc
     3121 gacacaaaat ttattctaaa tgcataataa atactgataa catcttatag tttgtattat
     3181 attttgtatt atcgttgaca tgtataattt tgatatcaaa aactgatttt ccctttatta
     3241 ttttcgagat ttattttctt aattctcttt aacaaactag aaatattgta tatacaaaaa
     3301 atcataaata atagatgaat agtttaatta taggtgttca tcaatcgaaa aagcaacgta
     3361 tcttatttaa agtgcgttgc ttttttctca tttataaggt taaataattc tcatatatca
     3421 agcaaagtga caggcgccct taaatattct gacaaatgct ctttccctaa actcccccca
     3481 taaaaaaacc cgccgaagcg ggtttttacg ttatttgcgg attaacgatt actcgttatc
     3541 agaaccgccc agggggcccg agcttaagac tggccgtcgt tttacaacac agaaagagtt
     3601 tgtagaaacg caaaaaggcc atccgtcagg ggccttctgc ttagtttgat gcctggcagt
     3661 tccctactct cgccttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3721 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3781 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3841 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3901 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3961 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4021 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4081 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4141 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4201 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4261 ttgaagtggt gggctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4321 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4381 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4441 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga cgcgcgcgta
     4501 actcacgtta agggattttg gtcatgagct tgcgccgtcc cgtcaagtca gcgtaatgct
     4561 ctgctttt
//
