LOCUS       sequence-166664-        6166 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 6166)
  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 6166)
  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..6166
                     /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..2176
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2197..2271
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2276..2295
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        2345..2613
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2634..2708
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2713..2732
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(2834..2851)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3005..3593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3085..3104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3764..4555,4556..4624))
                     /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     4387..4406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4625..4729)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4756..6098
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5719..5766)
                     /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     6139..6157
                     /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 gtgcaggtag ggaacaaaag ctggagcttc tttgaaaaga
     1921 taatgtatga ttatgctttc actcatattt atacagaaac ttgatgtttt ctttagagta
     1981 tatacaaggt gattacatgt acgtttgaag tacaactcta gattttgtag tgccctcttg
     2041 ggctagcggt aaaggtgcgc attttttcac accctacaat gttctgttca aaagattttg
     2101 gtcaaacgct gtagaagtaa agttggtgcg catgtttcgg cgttcgaaac ttctccgcag
     2161 tgaaagataa atgatctgag aatactgttg taaaacgttt tagagctaga aatagcaagt
     2221 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt ggtgcttttt
     2281 ttgtttttta tgtcttcgag tcatgtaatt agttaatctg tcatagggaa caaaagctgg
     2341 agcttctttg aaaagataat gtatgattat gctttcactc atatttatac agaaacttga
     2401 tgttttcttt cgagtatata caaggtgatt acatgtacgt ttgaagtaca actctagatt
     2461 ttgtagtgcc ctcttgggct agcggtaaat gtgcgcattt tttcacaccc tacaatgttc
     2521 tgttcaaaag attttggtca aacgctgtag aagtgaaagt tggtgcgcat gtttcggcgt
     2581 tcgaaacttc tccgcagtga aagataaatg atcgcttcaa gaattgagta aacgttttag
     2641 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     2701 agtcggtggt gctttttttg ttttttatgt cttcgagtca tgtaattagt taatcgcgtg
     2761 cattccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2821 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2881 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2941 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3001 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3061 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3121 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3181 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3241 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3301 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3361 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3421 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     3481 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3541 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3601 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3661 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3721 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3781 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3841 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3901 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3961 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4021 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4081 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4141 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4201 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4261 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4321 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4381 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4441 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4501 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4561 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4621 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4681 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4741 gaaaagtgcc acctgaacga agcatctgtg cttcattttg tagaacaaaa atgcaacgcg
     4801 agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac
     4861 gcgaaagcgc tattttacca acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca
     4921 acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat
     4981 gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg ttctacaaaa
     5041 atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt tttctccttt
     5101 gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt taaggttaga
     5161 agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc cacttcccgc
     5221 gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc atccccgatt
     5281 atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata gcgttgatga
     5341 ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat atactacgta
     5401 taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt tcttactaca
     5461 atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag gtcgagttta
     5521 gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat agcacagaga
     5581 tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc aatattttag
     5641 tagctcgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca gagcgctttt
     5701 ggttttcaaa agcgctctga agttcctata ctttctagag aataggaact tcggaatagg
     5761 aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc tgcgcacata
     5821 cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata tatacatgag
     5881 aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct atttatgtag
     5941 gatgaaaggt agtctagtac ctcctgtgat attatcccat tccatgcggg gtatcgtatg
     6001 cttccttcag cactaccctt tagctgttct atatgctgcc actcctcaat tggattagtc
     6061 tcatccttca atgctatcat ttcctttgat attggatcat actaagaaac cattattatc
     6121 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
