LOCUS       sequence-166663-        6167 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at 
            sites X-4, XI-3, and XII-5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6167)
  AUTHORS   Jessop-Fabre MM, Jakociunas T, Stovicek V, Dai Z, Jensen MK, 
            Keasling JD, Borodina I
  TITLE     EasyClone-MarkerFree: A vector toolkit for marker-less integration 
            of genes into Saccharomyces cerevisiae via CRISPR-Cas9.
  JOURNAL   Biotechnol J. 2016 Aug;11(8):1110-7. doi: 10.1002/biot.201600147. 
            Epub 2016 Jun 23.
  PUBMED    27166612
REFERENCE   2  (bases 1 to 6167)
  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"; doi: 
            "10.1002/biot.201600147"; journalName: "Biotechnol J"; date: 
            "2016-08"; volume: "11"; issue: "8"; pages: "1110-7"
COMMENT     Sequence Label: pCfB3052(gRNA X-4, XI-3, XII-5)
FEATURES             Location/Qualifiers
     source          1..6167
                     /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"
     promoter        1472..1740
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1761..1835
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1840..1859
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        1909..2177
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2198..2272
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2277..2296
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        2346..2614
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2635..2709
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2714..2733
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(2835..2852)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3006..3594)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3086..3105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3765..4556,4557..4625))
                     /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     4388..4407
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4626..4730)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4757..6099
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5720..5767)
                     /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     6140..6158
                     /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 tcgcgccatt cgccattcag
     1381 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgga
     1441 atgcgtgcga tagggaacaa aagctggagc ttctttgaaa agataatgta tgattatgct
     1501 ttcactcata tttatacaga aacttgatgt tttctttcga gtatatacaa ggtgattaca
     1561 tgtacgtttg aagtacaact ctagattttg tagtgccctc ttgggctagc ggtaaaggtg
     1621 cgcatttttt cacaccctac aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag
     1681 tgaaagttgg tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc
     1741 cgccattcaa gagcagcaac gttttagagc tagaaatagc aagttaaaat aaggctagtc
     1801 cgttatcaac ttgaaaaagt ggcaccgagt cggtggtgct ttttttgttt tttatgtctt
     1861 cgagtcatgt aattagttaa gtgcaggtag ggaacaaaag ctggagcttc tttgaaaaga
     1921 taatgtatga ttatgctttc actcatattt atacagaaac ttgatgtttt ctttcgagta
     1981 tatacaaggt gattacatgt acgtttgaag tacaactcta gattttgtag tgccctcttg
     2041 ggctagcggt aaaggtgcgc attttttcac accctacaat gttctgttca aaagattttg
     2101 gtcaaacgct gtagaagtga aagttggtgc gcatgtttcg gcgttcgaaa cttctccgca
     2161 gtgaaagata aatgatcata tgtctctaat tttggaagtt ttagagctag aaatagcaag
     2221 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tggtgctttt
     2281 tttgtttttt atgtcttcga gtcatgtaat tagttaatct gtcataggga acaaaagctg
     2341 gagcttcttt gaaaagataa tgtatgatta tgctttcact catatttata cagaaacttg
     2401 atgttttctt tcgagtatat acaaggtgat tacatgtacg tttgaagtac aactctagat
     2461 tttgtagtgc cctcttgggc tagcggtaaa ggtgcgcatt ttttcacacc ctacaatgtt
     2521 ctgttcaaaa gattttggtc aaacgctgta gaagtgaaag ttggtgcgca tgtttcggcg
     2581 ttcgaaactt ctccgcagtg aaagataaat gatcttgtca cagtgtcaca tcaggtttta
     2641 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     2701 gagtcggtgg tgcttttttt gttttttatg tcttcgagtc atgtaattag ttaatcgcgt
     2761 gcattccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2821 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2881 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2941 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3001 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3061 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3121 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3181 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3241 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3301 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3361 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3421 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     3481 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3541 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3601 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3661 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3721 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3781 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3841 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3901 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3961 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4021 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4081 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4141 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4201 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4261 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4321 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4381 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4441 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4501 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4561 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4621 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4681 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4741 cgaaaagtgc cacctgaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc
     4801 gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa
     4861 cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc
     4921 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa
     4981 tgcaacgcga gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa
     5041 aatgcatccc gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt
     5101 tgtgcgctct ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag
     5161 aagaaggcta ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg
     5221 cgtttactga ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat
     5281 tatattctat accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg
     5341 attcttcatt ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt
     5401 ataggaaatg tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac
     5461 aatttttttg tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt
     5521 agatgcaagt tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag
     5581 atatatagca aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta
     5641 gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt
     5701 tggttttcaa aagcgctctg aagttcctat actttctaga gaataggaac ttcggaatag
     5761 gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat
     5821 acagctcact gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga
     5881 gaagaacggc atagtgcgtg tttatgctta aatgcgtact tatatgcgtc tatttatgta
     5941 ggatgaaagg tagtctagta cctcctgtga tattatccca ttccatgcgg ggtatcgtat
     6001 gcttccttca gcactaccct ttagctgttc tatatgctgc cactcctcaa ttggattagt
     6061 ctcatccttc aatgctatca tttcctttga tattggatca tactaagaaa ccattattat
     6121 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
