LOCUS       sequence-166362-        5293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at 
            site X-2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5293)
  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 5293)
  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: pCfB3020(gRNA X-2)
FEATURES             Location/Qualifiers
     source          1..5293
                     /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"
     primer_bind     complement(1961..1978)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2132..2720)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2212..2231)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2891..3682,3683..3751))
                     /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     3514..3533
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3752..3856)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3883..5225
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4846..4893)
                     /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     5266..5284
                     /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 ctctcgaagt ggtcacgtgc gttttagagc tagaaatagc aagttaaaat aaggctagtc
     1801 cgttatcaac ttgaaaaagt ggcaccgagt cggtggtgct ttttttgttt tttatgtctt
     1861 cgagtcatgt aattagttaa tcgcgtgcat tccagctgca ttaatgaatc ggccaacgcg
     1921 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1981 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2041 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2101 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2161 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2221 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2281 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2341 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2401 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2461 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2521 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     2581 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2641 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2701 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2761 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2821 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     2881 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2941 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3001 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3061 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3121 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3181 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3241 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3301 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3361 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3421 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3481 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3541 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3601 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3661 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3721 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3781 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3841 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgaacgaagc atctgtgctt
     3901 cattttgtag aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc
     3961 tgcattttta cagaacagaa atgcaacgcg aaagcgctat tttaccaacg aagaatctgt
     4021 gcttcatttt tgtaaaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc
     4081 tgagctgcat ttttacagaa cagaaatgca acgcgagagc gctattttac caacaaagaa
     4141 tctatacttc ttttttgttc tacaaaaatg catcccgaga gcgctatttt tctaacaaag
     4201 catcttagat tacttttttt ctcctttgtg cgctctataa tgcagtctct tgataacttt
     4261 ttgcactgta ggtccgttaa ggttagaaga aggctacttt ggtgtctatt ttctcttcca
     4321 taaaaaaagc ctgactccac ttcccgcgtt tactgattac tagcgaagct gcgggtgcat
     4381 tttttcaaga taaaggcatc cccgattata ttctataccg atgtggattg cgcatacttt
     4441 gtgaacagaa agtgatagcg ttgatgattc ttcattggtc agaaaattat gaacggtttc
     4501 ttctattttg tctctatata ctacgtatag gaaatgttta cattttcgta ttgttttcga
     4561 ttcactctat gaatagttct tactacaatt tttttgtcta aagagtaata ctagagataa
     4621 acataaaaaa tgtagaggtc gagtttagat gcaagttcaa ggagcgaaag gtggatgggt
     4681 aggttatata gggatatagc acagagatat atagcaaaga gatacttttg agcaatgttt
     4741 gtggaagcgg tattcgcaat attttagtag ctcgttacag tccggtgcgt ttttggtttt
     4801 ttgaaagtgc gtcttcagag cgcttttggt tttcaaaagc gctctgaagt tcctatactt
     4861 tctagagaat aggaacttcg gaataggaac ttcaaagcgt ttccgaaaac gagcgcttcc
     4921 gaaaatgcaa cgcgagctgc gcacatacag ctcactgttc acgtcgcacc tatatctgcg
     4981 tgttgcctgt atatatatat acatgagaag aacggcatag tgcgtgttta tgcttaaatg
     5041 cgtacttata tgcgtctatt tatgtaggat gaaaggtagt ctagtacctc ctgtgatatt
     5101 atcccattcc atgcggggta tcgtatgctt ccttcagcac taccctttag ctgttctata
     5161 tgctgccact cctcaattgg attagtctca tccttcaatg ctatcatttc ctttgatatt
     5221 ggatcatact aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     5281 aggccctttc gtc
//
