LOCUS       sequence-318095-        5289 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at 
            site II-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: pCfB9336 (pgRNA_II-1_NatMX)
FEATURES             Location/Qualifiers
     source          1..5289
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..269
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        290..364
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      369..388
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(490..507)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(661..1249)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(741..760)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1420..2211,2212..2280))
                     /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     2043..2062
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2281..2385)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2412..3754
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(3375..3422)
                     /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     3795..3813
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3851..3873)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     gene            3997..5116
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        3997..4340
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             4341..4913
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTTTGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      4919..5116
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 tctttgaaaa gataatgtat gattatgctt tcactcatat ttatacagaa acttgatgtt
       61 ttctttcgag tatatacaag gtgattacat gtacgtttga agtacaactc tagattttgt
      121 agtgccctct tgggctagcg gtaaaggtgc gcattttttc acaccctaca atgttctgtt
      181 caaaagattt tggtcaaacg ctgtataagt gaaagttggt gcgcatgttt cggcgttcga
      241 aacttctccg cagtgaaaga taaatgatca actgctcagg gcggataacg ttttagagct
      301 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      361 ggtggtgctt tttttgtttt ttatgtcttc gagtcatgta attagttaat cgcgtgcatt
      421 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
      481 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
      541 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
      601 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      661 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      721 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      781 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      841 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      901 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      961 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1021 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1081 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     1141 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     1201 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1261 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1321 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     1381 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1441 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1501 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1561 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1621 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1681 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1741 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1801 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1861 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1921 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1981 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     2041 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     2101 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     2161 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     2221 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2281 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     2341 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2401 agtgccacct gaacgaagca tctgtgcttc attttgtaga acaaaaatgc aacgcgagag
     2461 cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga
     2521 aagcgctatt ttaccaacga agaatctgtg cttcattttt gtaaaacaaa aatgcaacgc
     2581 gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa
     2641 cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct acaaaaatgc
     2701 atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc tcctttgtgc
     2761 gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag gttagaagaa
     2821 ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact tcccgcgttt
     2881 actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc ccgattatat
     2941 tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt tgatgattct
     3001 tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac tacgtatagg
     3061 aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt actacaattt
     3121 ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg agtttagatg
     3181 caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca cagagatata
     3241 tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaata ttttagtagc
     3301 tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc gcttttggtt
     3361 ttcaaaagcg ctctgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact
     3421 tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg cacatacagc
     3481 tcactgttca cgtcgcacct atatctgcgt gttgcctgta tatatatata catgagaaga
     3541 acggcatagt gcgtgtttat gcttaaatgc gtacttatat gcgtctattt atgtaggatg
     3601 aaaggtagtc tagtacctcc tgtgatatta tcccattcca tgcggggtat cgtatgcttc
     3661 cttcagcact accctttagc tgttctatat gctgccactc ctcaattgga ttagtctcat
     3721 ccttcaatgc tatcatttcc tttgatattg gatcatacta agaaaccatt attatcatga
     3781 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     3841 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     3901 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg ctgcatttaa atgatctgtt
     3961 tagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca gaataccctc
     4021 cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag
     4081 cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga
     4141 agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc cctcacagac
     4201 gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca
     4261 ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag ttctcacatc
     4321 acatccgaac ataaacaacc atgggtacca ctcttgacga cacggcttac cggtaccgca
     4381 ccagtgtccc gggggacgcc gaggccatcg aggcactgga tgggtccttc accaccgaca
     4441 ccgttttccg cgtcaccacc accggggacg gcttcaccct gcgggaggtg ccggtggacc
     4501 cgcccctgac caaggtgttc cccgacgacg aatcggacga cgaatcggac gacggggagg
     4561 acggcgaccc ggactcccgg acgttcgtcg cgtacgggga cgacggcgac ctggcgggct
     4621 tcgtggtcgt ctcgtactcc ggctggaacc gccggctgac cgtcgaggac atcgaggtcg
     4681 ccccggagca ccgggggcac ggggtcgggc gcgcgttgat ggggctcgcg acggagttcg
     4741 cccgcgagcg gggcgccggg cacctctggc tggaggtcac caacgtcaac gcaccggcga
     4801 tccacgcgta ccggcggatg gggttcaccc tctgcggcct ggacaccgcc ctgtacgacg
     4861 gcaccgcctc ggacggcgag caggcgctct acatgagcat gccctgcccc taatcagtac
     4921 tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt
     4981 agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat
     5041 ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat
     5101 gctggtcgct atactgctgt cgattcgata ctaacgccgc catccagtgt cgaaaacgag
     5161 ctgcggccgc ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg
     5221 cgatcggtgc gggcctcttc gctattacgc cagctggaat gcgtgcgata gggaacaaaa
     5281 gctggagct
//
