LOCUS       sequence-318100-        5288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at 
            site XVI-1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5288)
  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 5288)
  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: pCfB9344 (pgRNA_XVI-1_NatMX)
FEATURES             Location/Qualifiers
     source          1..5288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..268
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        289..363
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      368..387
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(489..506)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(660..1248)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(740..759)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1419..2210,2211..2279))
                     /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     2042..2061
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2280..2384)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2411..3753
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(3374..3421)
                     /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     3794..3812
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3850..3872)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     gene            3996..5115
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        3996..4339
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             4340..4912
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTTTGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      4918..5115
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 tctttgaaaa gataatgtat gattatgctt tcactcatat ttatacagaa acttgatttt
       61 tctttcgagt atatacaagg tgattacatg tacgtttgaa gtacaactct agattttgta
      121 gtgccctctt gggctagcgg taaaggtgcg cattttttca caccctacaa tgttctgttc
      181 aaaagatttt ggtcaaacgc tgtagaagtg aaagttggtg cgcatgtttc ggcgttcgaa
      241 acttctccgc agtgaaagat aaatgatcca ccattcaagt taccgagagt tttagagcta
      301 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      361 gtggtgcttt ttttgttttt tatgtcttcg agtcatgtaa ttagttaatc gcgtgcattc
      421 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
      481 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
      541 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
      601 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      661 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      721 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      781 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
      841 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
      901 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
      961 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1021 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1081 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     1141 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1201 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1261 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1321 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     1381 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     1441 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     1501 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     1561 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     1621 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     1681 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     1741 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     1801 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     1861 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     1921 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     1981 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     2041 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     2101 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     2161 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     2221 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     2281 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     2341 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     2401 gtgccacctg aacgaagcat ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc
     2461 gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgaa
     2521 agcgctattt taccaacgaa gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg
     2581 agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac
     2641 gcgagagcgc tattttacca acaaagaatc tatacttctt ttttgttcta caaaaatgca
     2701 tcccgagagc gctatttttc taacaaagca tcttagatta ctttttttct cctttgtgcg
     2761 ctctataatg cagtctcttg ataacttttt gcactgtagg tccgttaagg ttagaagaag
     2821 gctactttgg tgtctatttt ctcttccata aaaaaagcct gactccactt cccgcgttta
     2881 ctgattacta gcgaagctgc gggtgcattt tttcaagata aaggcatccc cgattatatt
     2941 ctataccgat gtggattgcg catactttgt gaacagaaag tgatagcgtt gatgattctt
     3001 cattggtcag aaaattatga acggtttctt ctattttgtc tctatatact acgtatagga
     3061 aatgtttaca ttttcgtatt gttttcgatt cactctatga atagttctta ctacaatttt
     3121 tttgtctaaa gagtaatact agagataaac ataaaaaatg tagaggtcga gtttagatgc
     3181 aagttcaagg agcgaaaggt ggatgggtag gttatatagg gatatagcac agagatatat
     3241 agcaaagaga tacttttgag caatgtttgt ggaagcggta ttcgcaatat tttagtagct
     3301 cgttacagtc cggtgcgttt ttggtttttt gaaagtgcgt cttcagagcg cttttggttt
     3361 tcaaaagcgc tctgaagttc ctatactttc tagagaatag gaacttcgga ataggaactt
     3421 caaagcgttt ccgaaaacga gcgcttccga aaatgcaacg cgagctgcgc acatacagct
     3481 cactgttcac gtcgcaccta tatctgcgtg ttgcctgtat atatatatac atgagaagaa
     3541 cggcatagtg cgtgtttatg cttaaatgcg tacttatatg cgtctattta tgtaggatga
     3601 aaggtagtct agtacctcct gtgatattat cccattccat gcggggtatc gtatgcttcc
     3661 ttcagcacta ccctttagct gttctatatg ctgccactcc tcaattggat tagtctcatc
     3721 cttcaatgct atcatttcct ttgatattgg atcatactaa gaaaccatta ttatcatgac
     3781 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
     3841 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     3901 tgccgggagc agacaagccc gtcagggcgc gtcagcgggc tgcatttaaa tgatctgttt
     3961 agcttgcctc gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc
     4021 ttgacagtct tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc
     4081 ccatacatcc ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa
     4141 gcaaaaatta cggctcctcg ctgcagacct gcgagcaggg aaacgctccc ctcacagacg
     4201 cgttgaattg tccccacgcc gcgcccctgt agagaaatat aaaaggttag gatttgccac
     4261 tgaggttctt ctttcatata cttcctttta aaatcttgct aggatacagt tctcacatca
     4321 catccgaaca taaacaacca tgggtaccac tcttgacgac acggcttacc ggtaccgcac
     4381 cagtgtcccg ggggacgccg aggccatcga ggcactggat gggtccttca ccaccgacac
     4441 cgttttccgc gtcaccacca ccggggacgg cttcaccctg cgggaggtgc cggtggaccc
     4501 gcccctgacc aaggtgttcc ccgacgacga atcggacgac gaatcggacg acggggagga
     4561 cggcgacccg gactcccgga cgttcgtcgc gtacggggac gacggcgacc tggcgggctt
     4621 cgtggtcgtc tcgtactccg gctggaaccg ccggctgacc gtcgaggaca tcgaggtcgc
     4681 cccggagcac cgggggcacg gggtcgggcg cgcgttgatg gggctcgcga cggagttcgc
     4741 ccgcgagcgg ggcgccgggc acctctggct ggaggtcacc aacgtcaacg caccggcgat
     4801 ccacgcgtac cggcggatgg ggttcaccct ctgcggcctg gacaccgccc tgtacgacgg
     4861 caccgcctcg gacggcgagc aggcgctcta catgagcatg ccctgcccct aatcagtact
     4921 gacaataaaa agattcttgt tttcaagaac ttgtcatttg tatagttttt ttatattgta
     4981 gttgttctat tttaatcaaa tgttagcgtg atttatattt tttttcgcct cgacatcatc
     5041 tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg
     5101 ctggtcgcta tactgctgtc gattcgatac taacgccgcc atccagtgtc gaaaacgagc
     5161 tgcggccgcc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc
     5221 gatcggtgcg ggcctcttcg ctattacgcc agctggaatg cgtgcgatag ggaacaaaag
     5281 ctggagct
//
