LOCUS       sequence-318103-        4291 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 XV-1, (Chr XV: 
            93186..93205).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4291)
  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 4291)
  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: pCfB9362(XV-1_Markerfree_BackBone)
FEATURES             Location/Qualifiers
     source          1..4291
                     /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(1069..1234)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1155..1179
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      1293..1482
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    complement(2034..2067)
                     /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(2274..2293)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2299..2318)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2360..2451
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2452..2520,2521..3312)
                     /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(2670..2689)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3434..4022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3923..3942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4176..4193
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(4291,1..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 tattgattta accatgggtg ctatttaact tgacgaagaa
      601 gtactttgct tgcgtgtcta tgctgctgga atgactgcgt tttatttcca tctaaccact
      661 cgaaacatat agtaataatc attatgatat gtcattttta cgtacctaac ggaaacacct
      721 ggcattttta ctctatttgt cgatcgtatc acttcgcttt caagctaaaa tattaataaa
      781 cgcaccgggg tattagatat acaacccata gttttcttca tttcacctat atctttccgg
      841 tatctcaata gggcttttgc aaacaaggcc gaacctcaca ttgaaaaatt tcacggcgtt
      901 ctgcaatccc gatcataaga aatgtaaaaa aataaaaggc cgccagaacg gttttatata
      961 aatgtacggg ttgaacgaac gtcacatgtg tacggatgta gttgtgtcac ggtatcatgg
     1021 ttagagacac agtatatgaa gattgaaaac ggaagacgtg caacgcttga gcgacctcat
     1081 gctatacctg agaaagcaac ctgacctaca ggaaagagtt actcaagaat aagaattttc
     1141 gttttaaaac ctaagagtca ctttaaaatt tgtatacact tatttttttt ataacttatt
     1201 taataataaa aatcataaat cataagaaat tcgcttattt agaagtgtca acaacgtatc
     1261 taccaacgga atgcgtgcga tcgcgtgcat tcatccgctc taaccgaaaa ggaaggagtt
     1321 agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg
     1381 ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt
     1441 atactgaaaa ccttgcttga gaaggttttg ggacgctcga agagtggcct tctagcctat
     1501 tgatggtgta gcaaatattc tagtgttaac tttgactcac tttatgaact aaatgcgtta
     1561 ttgatcgcca gtaaaaatta tgtagtttga aagaaacgat tgaaagtgta tgtagtttat
     1621 gaattacaaa tgagtaacaa taaaaaagta aatatgaaat tttcatttca gttaacttac
     1681 accttgaaac ccttagatct tcttctacct ctagctcttt cgaactttct accggtggac
     1741 aagattcttg gagccttacc cttgtgtgga cccataccga agtgtctgac agcttctctg
     1801 gagtttcttg gacctctcaa gatcaaagtg ttttgaccct ttggagctct gacagctaat
     1861 tgatccaaag tgatacattc accaccagcc ttaacaatct tggctctggc accagcagtg
     1921 aatctcaaag cagcaacagt ggtctttggg aattcaaaga ttctggcatc gtcagtaaca
     1981 gtaccaacaa cgacatcgcg tgcattcgcg gccgcattta aatccccact tcagaagttc
     2041 ctatactttc tagagaatag gaacttcact atagagtcga ataagggcga caccccataa
     2101 ttagcccggg cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgcctggc
     2161 agttccctac tctcgcatgg ggagtcccca cactaccatc ggcgctacgg cgtttcactt
     2221 ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc aaacaagggg
     2281 tgttatgagc catattcagg tataaatggg ctcgcgataa tgttcagaat tggttaattg
     2341 gttgtaacac tgacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2401 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagaa tatgagtatt
     2461 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2521 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     2581 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     2641 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     2701 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2761 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2821 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2881 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2941 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagcg
     3001 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3061 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3121 ccggctggct ggtttattgc tgataaatcc ggagccggtg agcgtggttc tcgcggtatc
     3181 atcgcagcgc tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3241 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3301 aagcattggt aagcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa
     3361 atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga
     3421 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3481 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3541 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     3601 tagcccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3661 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3721 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3781 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3841 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3901 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3961 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4021 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4081 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4141 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4201 aaggcgagag tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc
     4261 tttttgcgtt tctacaaact ctttctgtgt t
//
