LOCUS       sequence-324887-        4441 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 VII-1, (Chr VII: 
            438509..438490).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4441)
  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 4441)
  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: pCfB9358(VII-1_Markerfree_BackBone)
FEATURES             Location/Qualifiers
     source          1..4441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(612..777)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     698..722
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      836..1025
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    complement(1623..1656)
                     /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(1863..1882)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1888..1907)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1949..2040
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2041..2109,2110..2901)
                     /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(2259..2278)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3023..3611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3512..3531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3765..3782
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3880..3897
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3881..3897
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4367..4400
                     /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 tctgcctggg tagattttgg cacctgaaaa agaatcgatt tcaaccttca ttttatcact
       61 atattaatcc acgtttttca aacatatttg aaaaaaaaaa tgttagtaag tctatgcttt
      121 tcataggtag ccatcttgtc aatataaata atggtaatcc gcatggttgt tcaggcgttc
      181 gcaaaattgt aggaagagga gaacgtggaa atatggactt tttgcaaatt cagaattcat
      241 tttactaggt tcttgcaaca tggataacat ctcacgctgt tatttttcat gcttctccat
      301 atcgtatata tgtaatgaag cattttcatt gggtttcctg ctcaaaggtt gacatatatt
      361 ataagttata ctggaacaag ccacaatcct gtcaaagtct aaattccggt gtattggcta
      421 tctgaagctc tctcatatat taatacattg ctttctagca caacgctgcc ctcggtcgcc
      481 attaattatg aatcatcatc atatagggac atacctctca agttattgtc ttggtgtctt
      541 gcttaacttg gcaatagcaa cagcgcaaac tgcgtaaacg tactaaaata cacaccatac
      601 gtgcaacgct tgagcgacct catgctatac ctgagaaagc aacctgacct acaggaaaga
      661 gttactcaag aataagaatt ttcgttttaa aacctaagag tcactttaaa atttgtatac
      721 acttattttt tttataactt atttaataat aaaaatcata aatcataaga aattcgctta
      781 tttagaagtg tcaacaacgt atctaccaac ggaatgcgtg cgatcgcgtg cattcatccg
      841 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta
      901 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca
      961 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct
     1021 cgaagagtgg cctcatgcag tacattgaca gtatatcact tctggatagg acgccaaccg
     1081 ccagtctgtg tggtatttcc tcccgtctct gcgtgccaga cgggatcagc ccgtctattc
     1141 tcgtgtcgtc ctgcctggag agactgaacc cccatctagc accgcagtaa ggatcattac
     1201 tttgtaggcg ggaaataaga ggttcaacaa ggaccaaaaa aaaaaacact aaaatctgag
     1261 atcaaaaata tgtggtgcac agatgtaacg ttccaaaatg tatggatggt aaggtctttt
     1321 tccaagaaac gtatcttttt ttcagctagg gccgcattta aatttttttt tttttcaatt
     1381 ttttttccta ttggagacgg aatggcaaag ctcacgctgg gcgatagcac gtaaccacgc
     1441 tggtgtattt tcatcctaat cattctcttt catctatttc gataagctac ttaatgtcga
     1501 agttttaact tccatttgtt ggaatgttca ttacagttta tacttactcc ttgctctttt
     1561 ataatataac caaacagacc ggaatcgcgt gcattcgcgg ccgcatttaa atccccactt
     1621 cagaagttcc tatactttct agagaatagg aacttcacta tagagtcgaa taagggcgac
     1681 accccataat tagcccgggc gaaaggccca gtctttcgac tgagcctttc gttttatttg
     1741 atgcctggca gttccctact ctcgcatggg gagtccccac actaccatcg gcgctacggc
     1801 gtttcacttc tgagttcggc atggggtcag gtgggaccac cgcgctactg ccgccaggca
     1861 aacaaggggt gttatgagcc atattcaggt ataaatgggc tcgcgataat gttcagaatt
     1921 ggttaattgg ttgtaacact gacccctatt tgtttatttt tctaaataca ttcaaatatg
     1981 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagaat
     2041 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     2101 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     2161 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2221 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     2281 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     2341 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     2401 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     2461 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     2521 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2581 cctgtagcga tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2641 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2701 tcggcccttc cggctggctg gtttattgct gataaatccg gagccggtga gcgtggttct
     2761 cgcggtatca tcgcagcgct ggggccagat ggtaagccct cccgtatcgt agttatctac
     2821 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2881 tcactgatta agcattggta agcagagcat tacgctgact tgacgggacg gcgcaagctc
     2941 atgaccaaaa tcccttaacg tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg
     3001 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3061 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     3181 agccgtagtt agcccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3661 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3721 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3781 aggaagcgga aggcgagagt agggaactgc caggcatcaa actaagcaga aggcccctga
     3841 cggatggcct ttttgcgttt ctacaaactc tttctgtgtt gtaaaacgac ggccagtctt
     3901 aagctcgggc cccctgggcg gttctgataa cgagtaatcg ttaatccgca aataacgtaa
     3961 aaacccgctt cggcgggttt ttttatgggg ggagtttagg gaaagagcat ttgtcagaat
     4021 atttaagggc gcctgtcact ttgcttgata tatgagaatt atttaacctt ataaatgaga
     4081 aaaaagcaac gcactttaaa taagatacgt tgctttttcg attgatgaac acctataatt
     4141 aaactattca tctattattt atgatttttt gtatatacaa tatttctagt ttgttaaaga
     4201 gaattaagaa aataaatctc gaaaataata aagggaaaat cagtttttga tatcaaaatt
     4261 atacatgtca acgataatac aaaatataat acaaactata agatgttatc agtatttatt
     4321 atgcatttag aataaatttt gtgtcgccct tattcgactc actatagaag ttcctattct
     4381 ctagaaagta taggaacttc acttcatttt atttaaattt gcggccgcga atgcgtgcga
     4441 t
//
