LOCUS       sequence-318102-        4364 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 XIII-1, (Chr XIII: 
            306804..306823).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4364)
  AUTHORS   Babaei M, Sartori L, Karpukhin A, Abashkin D, Matrosova E, Borodina 
            I
  TITLE     Expansion of EasyClone-MarkerFree toolkit for Saccharomyces 
            cerevisiae genome with new integration sites.
  JOURNAL   FEMS Yeast Res. 2021 May 11;21(4). pii: 6249452. doi: 
            10.1093/femsyr/foab027.
  PUBMED    33893795
REFERENCE   2  (bases 1 to 4364)
  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"; journalName: "FEMS Yeast 
            Res."; date: "2021-05-11"; volume: "21(4). pii: 6249452. doi"; 
            pages: " 10.1093/femsyr/foab027"
COMMENT     Sequence Label: pCfB9361(XIII-1_Markerfree_BackBone)
FEATURES             Location/Qualifiers
     source          1..4364
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    487..520
                     /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(1112..1277)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1198..1222
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      1336..1525
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    complement(2107..2140)
                     /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(2347..2366)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2372..2391)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2433..2524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2525..2593,2594..3385)
                     /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"
     primer_bind     complement(2743..2762)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3507..4095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3996..4015
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4249..4266
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(4364,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccctgggcg gttctgataa cgagtaatcg
       61 ttaatccgca aataacgtaa aaacccgctt cggcgggttt ttttatgggg ggagtttagg
      121 gaaagagcat ttgtcagaat atttaagggc gcctgtcact ttgcttgata tatgagaatt
      181 atttaacctt ataaatgaga aaaaagcaac gcactttaaa taagatacgt tgctttttcg
      241 attgatgaac acctataatt aaactattca tctattattt atgatttttt gtatatacaa
      301 tatttctagt ttgttaaaga gaattaagaa aataaatctc gaaaataata aagggaaaat
      361 cagtttttga tatcaaaatt atacatgtca acgataatac aaaatataat acaaactata
      421 agatgttatc agtatttatt atgcatttag aataaatttt gtgtcgccct tattcgactc
      481 actatagaag ttcctattct ctagaaagta taggaacttc acttcatttt atttaaattt
      541 gcggccgcga atgcgtgcga ttccttctac tgcggtttcc ctgcaggcac agctaaccgg
      601 agggaagaca agtcaagcgg acatgcagtt cccgctcagg cttctttgca gagaaatcgg
      661 aatatgaatt ttttgtgttt tttgttaact tctttaccat tcgcatcacg ggaagagtgt
      721 gttggcataa gaggcgcaag aaaaaggtga cccacggtgg tgggaaagag aagaaaaagc
      781 cgtttgcgga aagcaatgtt ttgcccattc agaaacaata tctttgctgt atccctacat
      841 gtattatgcc gcgttgcttg tgcaggtacg gagtccctgc acgatttttc aacaaggtct
      901 agatttgcta agacgatgag gcggagcccg aaacaaacaa gccaaaaacc ggaatttcgc
      961 gcttggtgtg ctttcgcatc ttttcgtgtg gtgaatgcac ctggtgcacg ctgcgaaggt
     1021 gacgtagcaa tccaaaaaag ggaagtctag gcgctttcaa ccgcccccca ccgtgcatgc
     1081 ggattggttg tcacaattac gtgcaacgct tgagcgacct catgctatac ctgagaaagc
     1141 aacctgacct acaggaaaga gttactcaag aataagaatt ttcgttttaa aacctaagag
     1201 tcactttaaa atttgtatac acttattttt tttataactt atttaataat aaaaatcata
     1261 aatcataaga aattcgctta tttagaagtg tcaacaacgt atctaccaac ggaatgcgtg
     1321 cgatcgcgtg cattcatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta
     1381 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat
     1441 ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct
     1501 tgagaaggtt ttgggacgct cgaagagtgg cctcaatgtt tcctcccctc ctgttgatgc
     1561 gggtaagggg tgtctgttat gcaggaacag gaaggtaaaa attaaatggg ccttaattct
     1621 gtagacactc tcttggggta ataaggttaa aagccattgt tgaagtacgg aatcatctct
     1681 tcattttttc agcgcttgta aggaattaat ccccaggaag gtgccgatgt aatgagagaa
     1741 gtattcttgt tgtatctctt tggttcggag tatttccttc tttcttttct ttgtagtttt
     1801 tatgcaaatg cagggaacag agtacccggt taaggctgtt agtcagccgc actctacaca
     1861 aaattacttg gcaaacacgt tggttgggaa aagctttata cttggcggtg tagtaattgt
     1921 aaaggatcaa gattgtgtca tgctaatgtg ccgctgtgta acaacctttt tgaagcgcgt
     1981 atttaccacc ggcaaaaggc cattgtgtca cataaaaaaa aactcccgcc aaagccaggt
     2041 tcattttgca taagattcac cacagtaatc gcgtgcattc gcggccgcat ttaaatcccc
     2101 acttcagaag ttcctatact ttctagagaa taggaacttc actatagagt cgaataaggg
     2161 cgacacccca taattagccc gggcgaaagg cccagtcttt cgactgagcc tttcgtttta
     2221 tttgatgcct ggcagttccc tactctcgca tggggagtcc ccacactacc atcggcgcta
     2281 cggcgtttca cttctgagtt cggcatgggg tcaggtggga ccaccgcgct actgccgcca
     2341 ggcaaacaag gggtgttatg agccatattc aggtataaat gggctcgcga taatgttcag
     2401 aattggttaa ttggttgtaa cactgacccc tatttgttta tttttctaaa tacattcaaa
     2461 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2521 gaatatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2581 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2641 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2701 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2761 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2821 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2881 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2941 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     3001 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     3061 gatgcctgta gcgatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     3121 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     3181 gcgctcggcc cttccggctg gctggtttat tgctgataaa tccggagccg gtgagcgtgg
     3241 ttctcgcggt atcatcgcag cgctggggcc agatggtaag ccctcccgta tcgtagttat
     3301 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3361 tgcctcactg attaagcatt ggtaagcaga gcattacgct gacttgacgg gacggcgcaa
     3421 gctcatgacc aaaatccctt aacgtgagtt acgcgcgcgt cgttccactg agcgtcagac
     3481 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3541 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3601 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     3661 gtgtagccgt agttagccca ccacttcaag aactctgtag caccgcctac atacctcgct
     3721 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3781 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3841 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3901 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3961 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4021 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4081 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4141 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4201 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4261 agcgaggaag cggaaggcga gagtagggaa ctgccaggca tcaaactaag cagaaggccc
     4321 ctgacggatg gcctttttgc gtttctacaa actctttctg tgtt
//
