LOCUS       sequence-318106-        4349 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 II-1, (Chr II: 
            341622..341641).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4349)
  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 4349)
  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: pCfB9355(II-1_Markerfree_BackBone)
FEATURES             Location/Qualifiers
     source          1..4349
                     /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(1120..1285)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1206..1230
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      1344..1533
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    complement(2092..2125)
                     /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(2332..2351)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2357..2376)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2418..2509
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2510..2578,2579..3370)
                     /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(2728..2747)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3492..4080
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3981..4000
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4234..4251
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(4349,1..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 taggtattaa aaagggacat acttttttta tatcttttcc
      601 tttggcgttc acagttactc ttttagaact ccttgttgtt gtgtaatgca tttacccatt
      661 cccgatataa ctaccacttt ggagaactgt agacaaagac cgctcttact gagtatacat
      721 tttactttcg tagaccgact tttaactcta ttaggaaaag caattgactt ttgaacttcg
      781 atgacgattg tgcaaaatac aaatcgtttc aagcggtgtt cggaatagct cacagaaatg
      841 gcgaagatat gttgaaaaat acataccgat gtcatttgtg aaaaacaaaa acacaagcta
      901 caatttgtat atttactcaa ttacatatac caattgagta ataattaaac aagaaggcaa
      961 aaataaacta aagcaaaata gtataccatc tttggttcct ggaccacatt ttctctcaat
     1021 atttttgaga tgtttttctt tactttgttt taagtagtct ttccatctcc cttcaacgga
     1081 aacaagtgca cgcaacatgt tattttacgt gcaacgcttg agcgacctca tgctatacct
     1141 gagaaagcaa cctgacctac aggaaagagt tactcaagaa taagaatttt cgttttaaaa
     1201 cctaagagtc actttaaaat ttgtatacac ttattttttt tataacttat ttaataataa
     1261 aaatcataaa tcataagaaa ttcgcttatt tagaagtgtc aacaacgtat ctaccaacgg
     1321 aatgcgtgcg atcgcgtgca ttcatccgct ctaaccgaaa aggaaggagt tagacaacct
     1381 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata
     1441 tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa
     1501 accttgcttg agaaggtttt gggacgctcg aagagtggcc tctgagcagt tttcttcccc
     1561 gggtggcaac cattctccaa cattagacaa actttacaaa gaagacaccc tacccggttg
     1621 cctccaaagc ccttctttct taacgcgagt accgctttgg gaggcctcat cttggttgtg
     1681 tcctcgtatg gcagcatctg ccattgagcg aaccctttcc cgttttacgc gcatggccta
     1741 gaccgatctt tagcagacat gccgagtgaa tttacctaag atttctaagg actcttctgc
     1801 atgtttccga aaatcaccgg aaaaaccttc ggttttttta gacgaaaact aggaagttgc
     1861 ttagctttcc ttgtcgtctt caaatctata tgccatcgga agagaatcag aagagaagat
     1921 cacgacttaa gagctttgct gttaagatag caagaaagct tgccaatagg cagttatgta
     1981 acctagttta tctacacctt ctttgattaa cttagctatg taggccggtg tgtttgttca
     2041 tatcacggca taatcgcgtg cattcgcggc cgcatttaaa tccccacttc agaagttcct
     2101 atactttcta gagaatagga acttcactat agagtcgaat aagggcgaca ccccataatt
     2161 agcccgggcg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctggcag
     2221 ttccctactc tcgcatgggg agtccccaca ctaccatcgg cgctacggcg tttcacttct
     2281 gagttcggca tggggtcagg tgggaccacc gcgctactgc cgccaggcaa acaaggggtg
     2341 ttatgagcca tattcaggta taaatgggct cgcgataatg ttcagaattg gttaattggt
     2401 tgtaacactg acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2461 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagaata tgagtattca
     2521 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2581 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2641 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2701 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     2761 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2821 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2881 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2941 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3001 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcgat
     3061 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3121 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3181 ggctggctgg tttattgctg ataaatccgg agccggtgag cgtggttctc gcggtatcat
     3241 cgcagcgctg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3301 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3361 gcattggtaa gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat
     3421 cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc
     3481 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3541 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3601 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     3661 gcccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3721 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3781 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3841 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3901 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3961 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4021 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4081 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4141 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4201 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4261 ggcgagagta gggaactgcc aggcatcaaa ctaagcagaa ggcccctgac ggatggcctt
     4321 tttgcgtttc tacaaactct ttctgtgtt
//
