LOCUS       sequence-318101-        4332 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 IV-1, (Chr IV: 
            132921..132940).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4332)
  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 4332)
  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: pCfB9356(IV-1_Markerfree_BackBone)
FEATURES             Location/Qualifiers
     source          1..4332
                     /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(1098..1263)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1184..1208
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      1322..1511
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    complement(2075..2108)
                     /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(2315..2334)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2340..2359)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2401..2492
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2493..2561,2562..3353)
                     /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(2711..2730)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3475..4063
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3964..3983
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4217..4234
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(4332,1..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 tatctctcca aggttagccg cccatataaa aaactctagg
      601 aaccaaattt ggtatactgc gtgcgcttga gattcagttt ttgtgtaaat cggttgctgc
      661 aaactattta tattaatgtt acatcgatgc aagattttag ccatgttcct agtttcttgt
      721 ctatgagcgt ttactcaacc gagactcttg tttgctatta tctcgattcg acgaacaaac
      781 agttccctgg gtattcgaac aaagcacagt tggtgatttt gtttgaaact acagacgtgc
      841 tctcttttac aaaataaatt gtaggaagtg atccgcacag gcagagggaa ttacagatca
      901 cgctcctctc ttatgactaa tgcactcagc attttctctt ccttataaac tccgtccaca
      961 gtacccgatc agctggctca tcgttattct aaattaactg atgactgatg ttacgtctgc
     1021 gtcaaattcc gccggaatat tgcgtagttt tagtaagaaa aaaacaaatt ccgacgggaa
     1081 ctctacgtgc aacgcttgag cgacctcatg ctatacctga gaaagcaacc tgacctacag
     1141 gaaagagtta ctcaagaata agaattttcg ttttaaaacc taagagtcac tttaaaattt
     1201 gtatacactt atttttttta taacttattt aataataaaa atcataaatc ataagaaatt
     1261 cgcttattta gaagtgtcaa caacgtatct accaacggaa tgcgtgcgat cgcgtgcatt
     1321 catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt
     1381 tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc
     1441 tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg
     1501 gacgctcgaa gagtggcctt tactttgcta ggttgagaaa aagaattctt tagaggatca
     1561 cacgtagtaa aagaaaaaaa agatctcttc gcgccactcc cggtttctct gtggatacgg
     1621 cgttactagc gttgcaagtg gcaaaaaaat gagtaatgtc acaatggcct ggccttgtcc
     1681 cactatccct tattcatggt tgaaaaatat gaccaaaatt gctggggaat tttctcttcg
     1741 gtagtggacg acgacgacat tagagcaata ggcaataatt taatttgcgc tttaggttct
     1801 tgctgacgat gatgcgtgag taatattatt accaagtttt aagggcctca tcgctggcca
     1861 ttttttacac ctgcttaaat acccgatgat gatatatata aatgatattg gctgcttatc
     1921 gataaagtgt atttgatatg ttcccgttta gtttattctt gtttcttatc tacataacaa
     1981 acaaaggaag gaccctgtat tgttttccta aagatgactg ctgctaaacc aaatcatcgc
     2041 gtgcattcgc ggccgcattt aaatccccac ttcagaagtt cctatacttt ctagagaata
     2101 ggaacttcac tatagagtcg aataagggcg acaccccata attagcccgg gcgaaaggcc
     2161 cagtctttcg actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcatg
     2221 gggagtcccc acactaccat cggcgctacg gcgtttcact tctgagttcg gcatggggtc
     2281 aggtgggacc accgcgctac tgccgccagg caaacaaggg gtgttatgag ccatattcag
     2341 gtataaatgg gctcgcgata atgttcagaa ttggttaatt ggttgtaaca ctgaccccta
     2401 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2461 aaatgcttca ataatattga aaaaggaaga atatgagtat tcaacatttc cgtgtcgccc
     2521 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2581 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2641 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2701 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     2761 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2821 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2881 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2941 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3001 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc gatggcaaca acgttgcgca
     3061 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3121 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3181 ctgataaatc cggagccggt gagcgtggtt ctcgcggtat catcgcagcg ctggggccag
     3241 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3301 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taagcagagc
     3361 attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac
     3421 gcgcgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3481 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3541 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3601 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttagcccacc acttcaagaa
     3661 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3721 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3781 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3841 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3901 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3961 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4021 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4081 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4141 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4201 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaaggcgaga gtagggaact
     4261 gccaggcatc aaactaagca gaaggcccct gacggatggc ctttttgcgt ttctacaaac
     4321 tctttctgtg tt
//
