LOCUS       sequence-313173-        4324 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 VIII-1, (Chr VIII: 
            293615..293634).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4324)
  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 4324)
  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: pCfB9359(VIII-1_Markerfree_BackBone)
FEATURES             Location/Qualifiers
     source          1..4324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(525..690)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     611..635
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      749..938
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    complement(1517..1550)
                     /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(1757..1776)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1782..1801)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1843..1934
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1935..2003,2004..2795)
                     /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(2153..2172)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2919..3507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3408..3427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3661..3678
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3776..3793
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3777..3793
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4263..4296
                     /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)"
ORIGIN
        1 atcctttgcg ctgtctcttt gaagaaaaat caggagtgca agatatcgat taattccttg
       61 gaagttatga tggttagtct tagtttaact ctcttgaaga agggtttttt cagttggtca
      121 acactcttta gaggtaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
      181 aaaaagagaa ttcttcaggt aatttaccat gattctacgt ttttgcaatc aaaaatgaaa
      241 ataatccgag cgcatgcgca agcagtccct gccaggccgc ttcggaaaaa ctttcgaaac
      301 caatactcct tacatccgcc ttgaatttaa gacctataat tgtcgtctct tctgcttgcc
      361 aataagccct aaaaacctta caccctaatt tgcacaagaa aactacgaaa aaaatttttc
      421 ctcttttttc cgcagcacca atctcgccct gctatttttt tggttcggag aaaaatagtc
      481 ggtcgatatg aattgaagtt tcattgtggc cacgtgcaac gcttgagcga cctcatgcta
      541 tacctgagaa agcaacctga cctacaggaa agagttactc aagaataaga attttcgttt
      601 taaaacctaa gagtcacttt aaaatttgta tacacttatt ttttttataa cttatttaat
      661 aataaaaatc ataaatcata agaaattcgc ttatttagaa gtgtcaacaa cgtatctacc
      721 aacggaatgc gtgcgatcgc gtgcattcat ccgctctaac cgaaaaggaa ggagttagac
      781 aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat
      841 ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac
      901 tgaaaacctt gcttgagaag gttttgggac gctcgaagag tggcctaata acctccgcga
      961 atggaaaaaa agaactcgga atatcaaaaa ttccggctca aaggaaaaat tttgcttcag
     1021 gaggggggaa ttatattcca gatgggacct gcaaggattt ctgtgtgagg ttttctcgga
     1081 ggaataatag tttttccaaa aattactttt ggcaactacg tgaatataat cgactacgga
     1141 tatccttcga gaaggccata aaaagaaccg tgacagggcc aaacagaaaa ataatgaaga
     1201 caagtttgct ttttttctca ctgtatttga ggatcggaag ctttttgcac tgaaaatatt
     1261 ctgggttaac aaggtttaaa gaagcgaact gccacaaagt atatgagagt ataaccggac
     1321 ggtacctata ctttgaagtt ggaataaata cggggtagac aaaaaggatg tgtttctgta
     1381 tgtctgatga gaatgatgtc atcaaagaac gtaaacaaca gtaactgtct atattcttgc
     1441 tttttattat gcgctgttac agcaggtagc ggagtttatc gcgtgcattc gcggccgcat
     1501 ttaaatcccc acttcagaag ttcctatact ttctagagaa taggaacttc actatagagt
     1561 cgaataaggg cgacacccca taattagccc gggcgaaagg cccagtcttt cgactgagcc
     1621 tttcgtttta tttgatgcct ggcagttccc tactctcgca tggggagtcc ccacactacc
     1681 atcggcgcta cggcgtttca cttctgagtt cggcatgggg tcaggtggga ccaccgcgct
     1741 actgccgcca ggcaaacaag gggtgttatg agccatattc aggtataaat gggctcgcga
     1801 taatgttcag aattggttaa ttggttgtaa cactgacccc tatttgttta tttttctaaa
     1861 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1921 gaaaaaggaa gaatatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1981 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2041 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2101 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2161 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2221 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2281 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2341 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2401 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2461 gtgacaccac gatgcctgta gcgatggcaa caacgttgcg caaactatta actggcgaac
     2521 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2581 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tccggagccg
     2641 gtgagcgtgg ttctcgcggt atcatcgcag cgctggggcc agatggtaag ccctcccgta
     2701 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2761 ctgagatagg tgcctcactg attaagcatt ggtaaaagca gagcattacg ctgacttgac
     2821 gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgcgc gtcgttccac
     2881 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2941 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3001 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3061 actgttcttc tagtgtagcc gtagttagcc caccacttca agaactctgt agcaccgcct
     3121 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3181 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3241 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3301 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3361 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3421 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3481 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3541 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3601 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3661 agcgagtcag tgagcgagga agcggaaggc gagagtaggg aactgccagg catcaaacta
     3721 agcagaaggc ccctgacgga tggccttttt gcgtttctac aaactctttc tgtgttgtaa
     3781 aacgacggcc agtcttaagc tcgggccccc tgggcggttc tgataacgag taatcgttaa
     3841 tccgcaaata acgtaaaaac ccgcttcggc gggttttttt atggggggag tttagggaaa
     3901 gagcatttgt cagaatattt aagggcgcct gtcactttgc ttgatatatg agaattattt
     3961 aaccttataa atgagaaaaa agcaacgcac tttaaataag atacgttgct ttttcgattg
     4021 atgaacacct ataattaaac tattcatcta ttatttatga ttttttgtat atacaatatt
     4081 tctagtttgt taaagagaat taagaaaata aatctcgaaa ataataaagg gaaaatcagt
     4141 ttttgatatc aaaattatac atgtcaacga taatacaaaa tataatacaa actataagat
     4201 gttatcagta tttattatgc atttagaata aattttgtgt cgcccttatt cgactcacta
     4261 tagaagttcc tattctctag aaagtatagg aacttcactt cattttattt aaatttgcgg
     4321 ccgc
//
