LOCUS       sequence-325948-        5287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at 
            site IX-1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5287)
  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 5287)
  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: pCfB9341 (pgRNA_IX-1_NatMX)
FEATURES             Location/Qualifiers
     source          1..5287
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     gene            175..1294
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        175..518
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             519..1091
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTTTGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1097..1294
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_RNA        1755..1829
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1834..1853
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1955..1972)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2126..2714)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2206..2225)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2885..3676,3677..3745))
                     /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     3508..3527
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3746..3850)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3877..5219
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4840..4887)
                     /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     5260..5278
                     /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 tcagcgggct
      121 gcatttaaat gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg
      181 gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac
      241 tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt
      301 gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga
      361 aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata
      421 aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta
      481 ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaccact cttgacgaca
      541 cggcttaccg gtaccgcacc agtgtcccgg gggacgccga ggccatcgag gcactggatg
      601 ggtccttcac caccgacacc gttttccgcg tcaccaccac cggggacggc ttcaccctgc
      661 gggaggtgcc ggtggacccg cccctgacca aggtgttccc cgacgacgaa tcggacgacg
      721 aatcggacga cggggaggac ggcgacccgg actcccggac gttcgtcgcg tacggggacg
      781 acggcgacct ggcgggcttc gtggtcgtct cgtactccgg ctggaaccgc cggctgaccg
      841 tcgaggacat cgaggtcgcc ccggagcacc gggggcacgg ggtcgggcgc gcgttgatgg
      901 ggctcgcgac ggagttcgcc cgcgagcggg gcgccgggca cctctggctg gaggtcacca
      961 acgtcaacgc accggcgatc cacgcgtacc ggcggatggg gttcaccctc tgcggcctgg
     1021 acaccgccct gtacgacggc accgcctcgg acggcgagca ggcgctctac atgagcatgc
     1081 cctgccccta atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt
     1141 atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt
     1201 ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg
     1261 cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca
     1321 tccagtgtcg aaaacgagct gcggccgcct acagggcgcg tccattcgcc attcaggctg
     1381 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggaatgc
     1441 gtgcgatagg gaacaaaagc tggagcttct ttgaaaagat aatgtatgat tatgctttca
     1501 ctcatattta tacagaaact tgatttttct ttcgagtata tacaaggtga ttacatgtac
     1561 gtttgaagta caactctaga ttttgtagtg ccctcttggg ctagcggtaa aggtgcgcat
     1621 tttttcacac cctacaatgt tctgttcaaa agattttggt caaacgctgt agaagtgaaa
     1681 gttggtgcgc atgtttcggc gttcgaaact tctccgcagt gaaagataaa tgacatctta
     1741 aatgaaagac agaggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     1801 caacttgaaa aagtggcacc gagtcggtgg tgcttttttt gttttttatg tcttcgagtc
     1861 atgtaattag ttaatcgcgt gcattccagc tgcattaatg aatcggccaa cgcgcgggga
     1921 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1981 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2041 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2101 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2161 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2221 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2281 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2341 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2401 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2461 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2521 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2581 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2641 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2701 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2761 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2821 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2881 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2941 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3001 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3061 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3121 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3181 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3241 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3301 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3361 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3421 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3481 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3541 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3601 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3661 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3721 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3781 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3841 gggttccgcg cacatttccc cgaaaagtgc cacctgaacg aagcatctgt gcttcatttt
     3901 gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt
     3961 tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca
     4021 tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct
     4081 gcatttttac agaacagaaa tgcaacgcga gagcgctatt ttaccaacaa agaatctata
     4141 cttctttttt gttctacaaa aatgcatccc gagagcgcta tttttctaac aaagcatctt
     4201 agattacttt ttttctcctt tgtgcgctct ataatgcagt ctcttgataa ctttttgcac
     4261 tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc tattttctct tccataaaaa
     4321 aagcctgact ccacttcccg cgtttactga ttactagcga agctgcgggt gcattttttc
     4381 aagataaagg catccccgat tatattctat accgatgtgg attgcgcata ctttgtgaac
     4441 agaaagtgat agcgttgatg attcttcatt ggtcagaaaa ttatgaacgg tttcttctat
     4501 tttgtctcta tatactacgt ataggaaatg tttacatttt cgtattgttt tcgattcact
     4561 ctatgaatag ttcttactac aatttttttg tctaaagagt aatactagag ataaacataa
     4621 aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg aaaggtggat gggtaggtta
     4681 tatagggata tagcacagag atatatagca aagagatact tttgagcaat gtttgtggaa
     4741 gcggtattcg caatatttta gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa
     4801 gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg aagttcctat actttctaga
     4861 gaataggaac ttcggaatag gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat
     4921 gcaacgcgag ctgcgcacat acagctcact gttcacgtcg cacctatatc tgcgtgttgc
     4981 ctgtatatat atatacatga gaagaacggc atagtgcgtg tttatgctta aatgcgtact
     5041 tatatgcgtc tatttatgta ggatgaaagg tagtctagta cctcctgtga tattatccca
     5101 ttccatgcgg ggtatcgtat gcttccttca gcactaccct ttagctgttc tatatgctgc
     5161 cactcctcaa ttggattagt ctcatccttc aatgctatca tttcctttga tattggatca
     5221 tactaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     5281 tttcgtc
//
