LOCUS       sequence-313182-        5289 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at 
            site VII-1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5289)
  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 5289)
  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: pCfB9339 (pgRNA_VII-1_NatMX)
FEATURES             Location/Qualifiers
     source          1..5289
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     gene            171..1290
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        171..514
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             515..1087
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1093..1290
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        1468..1736
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1757..1831
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1836..1855
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1957..1974)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2128..2716)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2208..2227)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2887..3678,3679..3747))
                     /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     3510..3529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3748..3852)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3879..5221
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4842..4889)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     5262..5280
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggat
      121 ttaaatgatc tgtttagctt gcctcgtccc cgccgggtca cccggccagc gacatggagg
      181 cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc
      241 gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata cattttgatg
      301 gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag cagggaaacg
      361 ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag
      421 gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat
      481 acagttctca catcacatcc gaacataaac aaccatgggt accactcttg acgacacggc
      541 ttaccggtac cgcaccagtg tcccggggga cgccgaggcc atcgaggcac tggatgggtc
      601 cttcaccacc gacaccgttt tccgcgtcac cgccaccggg gacggcttca ccctgcggga
      661 ggtgccggtg gacccgcccc tgaccaaggt gttccccgac gacgaatcgg acgacgaatc
      721 ggacgacggg gaggacggcg acccggactc ccggacgttc gtcgcgtacg gggacgacgg
      781 cgacctggcg ggcttcgtgg tcgtctcgta ctccggctgg aaccgccggc tgaccgtcga
      841 ggacatcgag gtcgccccgg agcaccgggg gcacggggtc gggcgcgcgt tgatggggct
      901 cgcgacggag ttcgcccgcg agcggggcgc cgggcacctc tggctggagg tcaccaacgt
      961 caacgcaccg gcgatccacg cgtaccggcg gatggggttc accctctgcg gcctggacac
     1021 cgccctgtac gacggcaccg cctcggacgg cgagcaggcg ctctacatga gcatgccctg
     1081 cccctaatca gtactgacaa taaaaagatt cttgttttca agaacttgtc atttgtatag
     1141 tttttttata ttgtagttgt tctattttaa tcaaatgtta gcgtgattta tatttttttt
     1201 cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc agaaagtaat atcatgcgtc
     1261 aatcgtatgt gaatgctggt cgctatactg ctgtcgattc gatactaacg ccgccatcca
     1321 gtgtcgaaaa cgagctgcgg ccgcctacag ggcgcgtcgc gccattcgcc attcaggctg
     1381 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggaatgc
     1441 gtgcgatagg gaacaaaagc tggagcttct ttgaaaagat aatgtatgat tatgctttca
     1501 ctcatattta tacagaaact tgatgttttc tttcgagtat atacaaggtg attacatgta
     1561 cgtttgaagt acaactctag attttgtagt gccctcttgg gctagcggta aaggtgcgca
     1621 ttttttcaca ccctacaatg ttctgttcaa aagattttgg tcaaacgctg tagaagtgaa
     1681 agttggtgcg catgtttcgg cgttcgaaac ttctccgcag tgaaagataa atgatcgtac
     1741 tgcatgatat attcaagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     1801 atcaacttga aaaagtggca ccgagtcggt ggtgcttttt ttgtttttta tgtcttcgag
     1861 tcatgtaatt agttaatcgc gtgcattcca gctgcattaa tgaatcggcc aacgcgcggg
     1921 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     1981 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2041 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2101 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2161 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2221 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2281 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2341 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2401 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2461 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2521 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     2581 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2641 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     2701 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     2761 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     2821 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     2881 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     2941 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3001 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3061 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3121 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3181 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3241 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3301 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3361 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3421 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3481 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3541 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3601 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3661 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3721 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     3781 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3841 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt
     3901 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca
     3961 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt
     4021 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag
     4081 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta
     4141 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc
     4201 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc
     4261 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa
     4321 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt
     4381 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga
     4441 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct
     4501 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca
     4561 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat
     4621 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt
     4681 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg
     4741 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga
     4801 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta
     4861 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa
     4921 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt
     4981 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta
     5041 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc
     5101 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct
     5161 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat
     5221 catactaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5281 cctttcgtc
//
