LOCUS       sequence-134842-        6145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at 
            sites X-2, XI-5, and XII-4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6145)
  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 6145)
  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: pCfB3053(gRNA X-2, XI-5, XII-4)
FEATURES             Location/Qualifiers
     source          1..6145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     gene            171..1290
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        171..514
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             515..1087
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1093..1290
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        1468..1736
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1757..1831
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1836..1855
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        1896..2164
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2185..2259
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2264..2283
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        2324..2592
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2613..2687
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2692..2711
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(2813..2830)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2984..3572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3064..3083)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3743..4534,4535..4603))
                     /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     4366..4385
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4604..4708)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4735..6077
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5698..5745)
                     /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     6118..6136
                     /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 tcagcgggat
      121 ttaaatgatc tgtttagctt gcctcgtccc cgccgggtca cccggccagc gacatggagg
      181 cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc
      241 gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata cattttgatg
      301 gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag cagggaaacg
      361 ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag
      421 gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat
      481 acagttctca catcacatcc gaacataaac aaccatgggt accactcttg acgacacggc
      541 ttaccggtac cgcaccagtg tcccggggga cgccgaggcc atcgaggcac tggatgggtc
      601 cttcaccacc gacaccgttt tccgcgtcac cgccaccggg gacggcttca ccctgcggga
      661 ggtgccggtg gacccgcccc tgaccaaggt gttccccgac gacgaatcgg acgacgaatc
      721 ggacgacggg gaggacggcg acccggactc ccggacgttc gtcgcgtacg gggacgacgg
      781 cgacctggcg ggcttcgtgg tcgtctcgta ctccggctgg aaccgccggc tgaccgtcga
      841 ggacatcgag gtcgccccgg agcaccgggg gcacggggtc gggcgcgcgt tgatggggct
      901 cgcgacggag ttcgcccgcg agcggggcgc cgggcacctc tggctggagg tcaccaacgt
      961 caacgcaccg gcgatccacg cgtaccggcg gatggggttc accctctgcg gcctggacac
     1021 cgccctgtac gacggcaccg cctcggacgg cgagcaggcg ctctacatga gcatgccctg
     1081 cccctaatca gtactgacaa taaaaagatt cttgttttca agaacttgtc atttgtatag
     1141 tttttttata ttgtagttgt tctattttaa tcaaatgtta gcgtgattta tatttttttt
     1201 cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc agaaagtaat atcatgcgtc
     1261 aatcgtatgt gaatgctggt cgctatactg ctgtcgattc gatactaacg ccgccatcca
     1321 gtgtcgaaaa cgagctgcgg ccgcctacag ggcgcgtcgc gccattcgcc attcaggctg
     1381 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggaatgc
     1441 gtgcgatagg gaacaaaagc tggagcttct ttgaaaagat aatgtatgat tatgctttca
     1501 ctcatattta tacagaaact tgatgttttc tttcgagtat atacaaggtg attacatgta
     1561 cgtttgaagt acaactctag attttgtagt gccctcttgg gctagcggta aaggtgcgca
     1621 ttttttcaca ccctacaatg ttctgttcaa aagattttgg tcaaacgctg tagaagtgaa
     1681 agttggtgcg catgtttcgg cgttcgaaac ttctccgcag tgaaagataa atgatcctct
     1741 cgaagtggtc acgtgcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     1801 atcaacttga aaaagtggca ccgagtcggt ggtgcttttt ttgtttttta tgtcttcgag
     1861 tcatgtaatt agttaaggga acaaaagctg gagcttcttt gaaaagataa tgtatgatta
     1921 tgctttcact catatttata cagaaacttg atgttttctt tcgagtatat acaaggtgat
     1981 tacatgtacg tttgaagtac aactctagat tttgtagtgc cctcttgggc tagcggtaaa
     2041 ggtgcgcatt ttttcacacc ctacaatgtt ctgttcaaaa gattttggtc aaacgctgta
     2101 gaagtgaaag ttggtgcgca tgtttcggcg ttcgaaactt ctccgcagtg aaagataaat
     2161 gatctgagaa tactgttgta aaacgtttta gagctagaaa tagcaagtta aaataaggct
     2221 agtccgttat caacttgaaa aagtggcacc gagtcggtgg tgcttttttt gttttttatg
     2281 tcttcgagtc atgtaattag ttaagggaac aaaagctgga gcttctttga aaagataatg
     2341 tatgattatg ctttcactca tatttataca gaaacttgat gttttctttc gagtatatac
     2401 aaggtgatta catgtacgtt tgaagtacaa ctctagattt tgtagtgccc tcttgggcta
     2461 gcggtaaagg tgcgcatttt ttcacaccct acaatgttct gttcaaaaga ttttggtcaa
     2521 acgctgtaga agtgaaagtt ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa
     2581 agataaatga tcgcttcaag aattgagtaa acgttttaga gctagaaata gcaagttaaa
     2641 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtggtg ctttttttgt
     2701 tttttatgtc ttcgagtcat gtaattagtt aatcgcgtgc attccagctg cattaatgaa
     2761 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     2821 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2881 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2941 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3001 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3061 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3121 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3181 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3241 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3301 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3361 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3421 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3481 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3541 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3601 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3661 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3721 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3781 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3841 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3901 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3961 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4021 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4081 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4141 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4201 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4261 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4321 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4381 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4441 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4501 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4561 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4621 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4681 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaacgaa
     4741 gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca
     4801 aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct attttaccaa
     4861 cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc
     4921 aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaga gcgctatttt
     4981 accaacaaag aatctatact tcttttttgt tctacaaaaa tgcatcccga gagcgctatt
     5041 tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat aatgcagtct
     5101 cttgataact ttttgcactg taggtccgtt aaggttagaa gaaggctact ttggtgtcta
     5161 ttttctcttc cataaaaaaa gcctgactcc acttcccgcg tttactgatt actagcgaag
     5221 ctgcgggtgc attttttcaa gataaaggca tccccgatta tattctatac cgatgtggat
     5281 tgcgcatact ttgtgaacag aaagtgatag cgttgatgat tcttcattgg tcagaaaatt
     5341 atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt tacattttcg
     5401 tattgttttc gattcactct atgaatagtt cttactacaa tttttttgtc taaagagtaa
     5461 tactagagat aaacataaaa aatgtagagg tcgagtttag atgcaagttc aaggagcgaa
     5521 aggtggatgg gtaggttata tagggatata gcacagagat atatagcaaa gagatacttt
     5581 tgagcaatgt ttgtggaagc ggtattcgca atattttagt agctcgttac agtccggtgc
     5641 gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa
     5701 gttcctatac tttctagaga ataggaactt cggaatagga acttcaaagc gtttccgaaa
     5761 acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac agctcactgt tcacgtcgca
     5821 cctatatctg cgtgttgcct gtatatatat atacatgaga agaacggcat agtgcgtgtt
     5881 tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg atgaaaggta gtctagtacc
     5941 tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc actacccttt
     6001 agctgttcta tatgctgcca ctcctcaatt ggattagtct catccttcaa tgctatcatt
     6061 tcctttgata ttggatcata ctaagaaacc attattatca tgacattaac ctataaaaat
     6121 aggcgtatca cgaggccctt tcgtc
//
