LOCUS       sequence-318109-        4314 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 XVI-1, (Chr XVI: 
            406267..406286).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4314)
  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 4314)
  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: pCfB9363(XVI-1_Markerfree_BackBone)
FEATURES             Location/Qualifiers
     source          1..4314
                     /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(1110..1275)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1196..1220
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      1334..1523
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    complement(2057..2090)
                     /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(2297..2316)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2322..2341)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2383..2474
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2475..2543,2544..3335)
                     /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(2693..2712)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3457..4045
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3946..3965
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4199..4216
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(4314,1..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 tttagccttg aaggtcgaag atttcttgga aagaagattg
      601 caaactcaag tctacaagct aggtttagcc aagtctgtcc accatgcaag agttttaatc
      661 actcaaaggc acattgctgt tggtaagcaa attgtcaaca tcccctcttt catggtcaga
      721 ttggactctg agaagcacat tgactttgct ccaacatctc cattcggtgg tgctagacca
      781 ggcagagttg ctagaagaaa tgctgcaagg aaggcggaag cttctggtga agctgctgat
      841 gaagctgatg aggccgatga agaataattg tgctgatttt tatttcttaa ttacgtgttg
      901 aaatcacgat actggcatcc gataggggag ttgtattcac aatactatcg attttgtaaa
      961 ataggacgaa gaacttttta tatacgagca tttcctaatt agtaggaagc ggaaaataat
     1021 aatataagaa agtaaacgca aaagataggc tgactgcctt cattcgacta ggaggtgagg
     1081 cgacatattt gtcaccacgt gcaacgcttg agcgacctca tgctatacct gagaaagcaa
     1141 cctgacctac aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc
     1201 actttaaaat ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa
     1261 tcataagaaa ttcgcttatt tagaagtgtc aacaacgtat ctaccaacgg aatgcgtgcg
     1321 atcgcgtgca ttcatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg
     1381 tccctattta tttttttata gttatgttag tattaagaac gttatttata tttcaaattt
     1441 ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg
     1501 agaaggtttt gggacgctcg aagagtggcc ttagagaggt ggatggctcg ggtgagcttg
     1561 attgtacact gcagcaacga tgctttttct acccatttta tgaagtttaa catccgtacc
     1621 tttccacctc caaacatttt ttgtaacttc gtcctttgaa aaatcaagaa gtaataggtg
     1681 tgcagtatag ggccgcttga gcgcgcaata tcggtgagtg aggtaagatc catccatacc
     1741 ttagcaaata tggtagtgag gaggccaact gtattgcgtt aaaggcaaaa ggattggtat
     1801 atacgaatga ttggtaattt gaaaagtagg tttcgaatca aagaaactga gacagtcaag
     1861 gacactaaac aaaaatgggt aaatcgtatg tccatataac ttcaaaatga aaatatagca
     1921 gttgaaacat atcaattaaa tcattataca tctccaataa acatgtatgc aagaggaaag
     1981 cgtaaatatc ttcgatttcg acaatacttt gctactgatc gcgtgcattc gcggccgcat
     2041 ttaaatcccc acttcagaag ttcctatact ttctagagaa taggaacttc actatagagt
     2101 cgaataaggg cgacacccca taattagccc gggcgaaagg cccagtcttt cgactgagcc
     2161 tttcgtttta tttgatgcct ggcagttccc tactctcgca tggggagtcc ccacactacc
     2221 atcggcgcta cggcgtttca cttctgagtt cggcatgggg tcaggtggga ccaccgcgct
     2281 actgccgcca ggcaaacaag gggtgttatg agccatattc aggtataaat gggctcgcga
     2341 taatgttcag aattggttaa ttggttgtaa cactgacccc tatttgttta tttttctaaa
     2401 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2461 gaaaaaggaa gaatatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2521 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2581 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2641 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2701 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2761 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2821 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2881 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2941 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3001 gtgacaccac gatgcctgta gcgatggcaa caacgttgcg caaactatta actggcgaac
     3061 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3121 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tccggagccg
     3181 gtgagcgtgg ttctcgcggt atcatcgcag cgctggggcc agatggtaag ccctcccgta
     3241 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3301 ctgagatagg tgcctcactg attaagcatt ggtaagcaga gcattacgct gacttgacgg
     3361 gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgcgcgt cgttccactg
     3421 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3481 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3541 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3601 tgttcttcta gtgtagccgt agttagccca ccacttcaag aactctgtag caccgcctac
     3661 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3721 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3781 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3841 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3901 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3961 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4021 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4081 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4141 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4201 cgagtcagtg agcgaggaag cggaaggcga gagtagggaa ctgccaggca tcaaactaag
     4261 cagaaggccc ctgacggatg gcctttttgc gtttctacaa actctttctg tgtt
//
