LOCUS       sequence-166357-        4552 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-5 (Chr XI: 
            11779..118967).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4552)
  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 4552)
  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: pCfB3037(XI-5 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4552
                     /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(2019..2184)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2105..2129
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      2243..2430
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2447..2466)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2448..2466)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2476..2492)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2485..2507)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(3050..3083)
                     /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(3553..3569)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3668..3685)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3839..4427)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3919..3938)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4551..4552,1..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 gtttaatgcg
     1321 gagaagtcgt tgatagcatt tccgaaggct tttccacatt gactgcagta tctaaaagag
     1381 ctccagcagc ggtcttagta gttcctgtgc ttgccgcttt gcagtataca gggacattgg
     1441 gaactacgga ggaaaaatcg gaatgctctg agctcttctc aaaaccagac gaactcgtct
     1501 tatttctcag taaattaccc aacatataaa ctctccctct tcaccttctc tacaaggcca
     1561 cacacaagac agttttaaca ggcaaatggt actggaggct ctcaccaatg gggtttaaga
     1621 cgctccaaac acacgctctc ctttttttct ttatatatcg gtagcaacga aagctagtcg
     1681 cagaacgtta cacactaaag cagatcaatc aatcaatcct gagaacctgc ttcgttcaaa
     1741 ccatccacta gaagaaacac gcacgtgtac ccaaaaatag agacaaacaa acagacaaaa
     1801 ataacaatga ttcctaccga ctaaagtaaa acaacactca cgtaataaaa aatcctagat
     1861 acaactcact tgtattcctc tcttctttga ccatcactaa agcttctctc ttactatatt
     1921 cctcaaataa aaaaaagttg caatgattac ttaccaatgt gccataaact ccgtgcacca
     1981 attaagtcct cagcgagctc gcatggaatg cgtgcgatga gcgacctcat gctatacctg
     2041 agaaagcaac ctgacctaca ggaaagagtt actcaagaat aagaattttc gttttaaaac
     2101 ctaagagtca ctttaaaatt tgtatacact tatttttttt ataacttatt taataataaa
     2161 aatcataaat cataagaaat tcgcttattt agaagtgtca acaacgtatc taccaacgga
     2221 atgcgtgcga tcgcgtgcat tcatccgctc taaccgaaaa ggaaggagtt agacaacctg
     2281 aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat
     2341 ttcaaatttt ctttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc
     2401 ttgcttgaga aggttttggg acgctcgaag atcgcgtccc aattcgccct atagtgagtc
     2461 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     2521 tacccctgca ggactagtgc tgaggcatta ctattggctg cttcatagta cacccaattg
     2581 cccacaaccc aagttaatca taagctcttg ttgtcgatga attgctcaaa atgtggccat
     2641 tctttcaatt ttgcacctct aaaactgctc cgtgggttgt gacaacaacc ttaagggtaa
     2701 tggacttttt tagaaagaca aatgagatga tttcacctac tggtatatca tttatttata
     2761 cacgaccgtt ttgacatttc agagttgcac gttggtatat cctaatattc tcacaagcat
     2821 cacagtgaag aaacgcaaga aaatggaaaa aattcccagg tggttgttgt cttctcttat
     2881 atcgtcggtg ctgtgcatac ttggggccct gtgtgtgccg ttgttatcgg ttgccttcga
     2941 tagcaagcgc aatagccaat ctaagttggt caactatggt ctttctctaa gtgccggatc
     3001 tatgatcatt aagacctcag cgcggccgca aatttaaata aaatgaagtg aagttcctat
     3061 actttctaga gaataggaac ttctatagtg agtcgaataa gggcgacaca aaatttattc
     3121 taaatgcata ataaatactg ataacatctt atagtttgta ttatattttg tattatcgtt
     3181 gacatgtata attttgatat caaaaactga ttttcccttt attattttcg agatttattt
     3241 tcttaattct ctttaacaaa ctagaaatat tgtatataca aaaaatcata aataatagat
     3301 gaatagttta attataggtg ttcatcaatc gaaaaagcaa cgtatcttat ttaaagtgcg
     3361 ttgctttttt ctcatttata aggttaaata attctcatat atcaagcaaa gtgacaggcg
     3421 cccttaaata ttctgacaaa tgctctttcc ctaaactccc cccataaaaa aacccgccga
     3481 agcgggtttt tacgttattt gcggattaac gattactcgt tatcagaacc gcccaggggg
     3541 cccgagctta agactggccg tcgttttaca acacagaaag agtttgtaga aacgcaaaaa
     3601 ggccatccgt caggggcctt ctgcttagtt tgatgcctgg cagttcccta ctctcgcctt
     3661 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3721 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3781 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3841 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3901 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3961 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4021 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4081 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4141 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4201 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtgggcta
     4261 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4321 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4381 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4441 tcttttctac ggggtctgac gctcagtgga acgacgcgcg cgtaactcac gttaagggat
     4501 tttggtcatg agcttgcgcc gtcccgtcaa gtcagcgtaa tgctctgctt tt
//
