LOCUS       sequence-166360-        4433 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 XII-4 (Chr 
            XII:830227..831248).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4433)
  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 4433)
  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: pCfB3040(XII-4 MarkerFree)
FEATURES             Location/Qualifiers
     source          1..4433
                     /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(1859..2024)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1945..1969
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      2083..2272
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2289..2308)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2290..2308)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2318..2334)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2327..2349)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2931..2964)
                     /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(3434..3450)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3549..3566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3720..4308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3800..3819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4432..4433,1..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 gtttaatgta
     1321 tccggctgtt ccttcatagc cctttcaatg aacgttgcag ccctttgaag attggccatt
     1381 ttgtcaggac tcgagcctga cagttggacc aacgcaactt taattttttg tgaaagaatc
     1441 ttcgaagcac tcatactggc gatcttcacg ccctcctgct attacaaaag ctgtgttttt
     1501 acaagaatca aattaagtta gcaagatatt atacaacatt attgataatt tcaatatcgt
     1561 gttcgtacct gatgacgtat ctgtgcattg ataaggcccg catggtttca gaaagcagag
     1621 cggaacgatt ccaaattagt ggccttgtgc tttgcatgtc aattgtgtta ccttcagctc
     1681 gtggatttgt tttatcaata cacagtctac agtcaagaat tttttttatc aaattttgcg
     1741 ttcgagcgta taaaatagcc gctgtagcta cttaagttcc tgttcagcga tagttttttt
     1801 ccatcacacg tactatggca attaagtcct cagcgagctc gcatggaatg cgtgcgatga
     1861 gcgacctcat gctatacctg agaaagcaac ctgacctaca ggaaagagtt actcaagaat
     1921 aagaattttc gttttaaaac ctaagagtca ctttaaaatt tgtatacact tatttttttt
     1981 ataacttatt taataataaa aatcataaat cataagaaat tcgcttattt agaagtgtca
     2041 acaacgtatc taccaacgga atgcgtgcga tcgcgtgcat tcatccgctc taaccgaaaa
     2101 ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt
     2161 attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc
     2221 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga agatcgcgtc
     2281 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     2341 gtgactggga aaaccctggc gttacccctg caggactagt gctgaggcat taatagttta
     2401 ctcaattctt gaagccaatt tgtacaattc cccattagag tcaaataaaa ggatgcctca
     2461 cggaggtatg ttacccgcgc tatttcacat ggctcattga attagaggtg gaatttggtg
     2521 taccctcccc tcctcatctg atgaagtagt gatccgacaa ttcttaaaag ttgtagacat
     2581 tacttttacc accaactaag ttgtatttat attgctaccc ttatcctttt atatctaact
     2641 agcgctcata aggttggggc aatactaaaa ctgtgttctt attcaactca ttaaatacgt
     2701 ggcagtacgt accctattag aaacaatagg aaacagcaga gtcggaagaa gccaaatgcc
     2761 agatttgaag tccaaaacct tgtcaagcca atctttggga gcggctattc ctccagaaat
     2821 tgtgtaccaa atacttacat accagtttag ggatttgtta agaaatgacc atccaggtac
     2881 ggcagaaaat taagacctca gcgcggccgc aaatttaaat aaaatgaagt gaagttccta
     2941 tactttctag agaataggaa cttctatagt gagtcgaata agggcgacac aaaatttatt
     3001 ctaaatgcat aataaatact gataacatct tatagtttgt attatatttt gtattatcgt
     3061 tgacatgtat aattttgata tcaaaaactg attttccctt tattattttc gagatttatt
     3121 ttcttaattc tctttaacaa actagaaata ttgtatatac aaaaaatcat aaataataga
     3181 tgaatagttt aattataggt gttcatcaat cgaaaaagca acgtatctta tttaaagtgc
     3241 gttgcttttt tctcatttat aaggttaaat aattctcata tatcaagcaa agtgacaggc
     3301 gcccttaaat attctgacaa atgctctttc cctaaactcc ccccataaaa aaacccgccg
     3361 aagcgggttt ttacgttatt tgcggattaa cgattactcg ttatcagaac cgcccagggg
     3421 gcccgagctt aagactggcc gtcgttttac aacacagaaa gagtttgtag aaacgcaaaa
     3481 aggccatccg tcaggggcct tctgcttagt ttgatgcctg gcagttccct actctcgcct
     3541 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3601 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3661 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3721 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3781 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3841 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3901 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3961 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4021 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4081 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtgggct
     4141 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4201 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4261 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4321 atcttttcta cggggtctga cgctcagtgg aacgacgcgc gcgtaactca cgttaaggga
     4381 ttttggtcat gagcttgcgc cgtcccgtca agtcagcgta atgctctgct ttt
//
