LOCUS       sequence-324895-        4359 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 4359)
  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 4359)
  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..4359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(562..727)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     648..672
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      786..975
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    complement(1554..1587)
                     /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(1794..1813)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1819..1838)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1880..1971
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1972..2040,2041..2832)
                     /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(2190..2209)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2954..3542
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3443..3462
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3696..3713
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3811..3828
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3812..3828
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4298..4331
                     /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 gaatgcgtgc gatttgactc aatgcaacag tgtcataatc ctttgcgctg tctctttgaa
       61 gaaaaatcag gagtgcaaga tatcgattaa ttccttggaa gttatgatgg ttagtcttag
      121 tttaactctc ttgaagaagg gttttttcag ttggtcaaca ctctttagag gtaaaaaaaa
      181 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aagagaattc ttcatgtaat
      241 ttaccatgat tctacgtttt tgcaatcaaa aatgaagata atccgagcgc atgcgcaagc
      301 agtccctgcc atgccgcttc ggaaaaactt tcgaaaccaa tactccttac atccgccttg
      361 aatttaagac ctataattgt cgtctcttct gcttgccaat aagccctaaa aaccttacac
      421 cctaatttgc acaagaaaac tacgaaaaaa atttttcctc ttttttccgc agcaccaatc
      481 tcgccctgct atttttttgg ttcggagaaa aatagtcggt cgatatgaat tgaagtttca
      541 ttgtggccac gtgcaacgct tgagcgacct catgctatac ctgagaaagc aacctgacct
      601 acaggaaaga gttactcaag aataagaatt ttcgttttaa aacctaagag tcactttaaa
      661 atttgtatac acttattttt tttataactt atttaataat aaaaatcata aatcataaga
      721 aattcgctta tttagaagtg tcaacaacgt atctaccaac ggaatgcgtg cgatcgcgtg
      781 cattcatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt
      841 tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt
      901 tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt
      961 ttgggacgct cgaagagtgg cctaataacc tccgcgaatg gaaaaaaaga actcggaata
     1021 tcaaaaattc cggctcaaag gaaaaatttt gcttcaggag gggggaatta tattccagat
     1081 gggacctgca aggatttctg tgtgaggttt tctcggagga ataatagttt ttccaaaaat
     1141 tacttttggc aactacgtga atataatcga ctacggatat ccttcgagaa ggccataaaa
     1201 agaaccgtga cagggccaaa cagaaaaata atgaagacaa gtttgctttt tttctcactg
     1261 tatttgagga tcggaagctt tttgcactga aaatattctg ggttaacaag gtttaaagaa
     1321 gcgaactgcc acaaagtata tgagagtata accggacggt acctatactt tgaagttgga
     1381 ataaatacgg ggtagacaaa aaggatgtgt ttctgtatgt ctgatgagaa tgatgtcatc
     1441 aaagaacgta aacaacagta actgtctata ttcttgcttt ttattatgcg ctgttacagc
     1501 aggtagcgga gtttatcgcg tgcattcgcg gccgcattta aatccccact tcagaagttc
     1561 ctatactttc tagagaatag gaacttcact atagagtcga ataagggcga caccccataa
     1621 ttagcccggg cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgcctggc
     1681 agttccctac tctcgcatgg ggagtcccca cactaccatc ggcgctacgg cgtttcactt
     1741 ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc aaacaagggg
     1801 tgttatgagc catattcagg tataaatggg ctcgcgataa tgttcagaat tggttaattg
     1861 gttgtaacac tgacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1921 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagaa tatgagtatt
     1981 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2041 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     2101 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     2161 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     2221 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2281 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2341 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2401 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2461 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagcg
     2521 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2581 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2641 ccggctggct ggtttattgc tgataaatcc ggagccggtg agcgtggttc tcgcggtatc
     2701 atcgcagcgc tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2761 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     2821 aagcattggt aagcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa
     2881 atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga
     2941 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3001 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3061 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     3121 tagcccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3181 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3241 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3301 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3361 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3421 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3481 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3541 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3601 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3661 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3721 aaggcgagag tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc
     3781 tttttgcgtt tctacaaact ctttctgtgt tgtaaaacga cggccagtct taagctcggg
     3841 ccccctgggc ggttctgata acgagtaatc gttaatccgc aaataacgta aaaacccgct
     3901 tcggcgggtt tttttatggg gggagtttag ggaaagagca tttgtcagaa tatttaaggg
     3961 cgcctgtcac tttgcttgat atatgagaat tatttaacct tataaatgag aaaaaagcaa
     4021 cgcactttaa ataagatacg ttgcttttta gattgatgaa cacctataat taaactattc
     4081 atctattatt tatgattttt tgtatataca atatttctag tttgttaaag agaattaaga
     4141 aaataaatct cgaaaataat aaagggaaaa tcagtttttg atatcaaaat tatacatgtc
     4201 aacgataata caaaatataa tacaaactat aagatgttat cagtatttat tatgcattta
     4261 gaataaattt tgtgtcgccc ttattcgact cactatagaa gttcctattc tctagaaagt
     4321 ataggaactt cacttcattt tatttaaatt tgcggccgc
//
