LOCUS       sequence-166366-        5285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at 
            site XI-2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5285)
  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 5285)
  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: pCfB3044(gRNA XI-2)
FEATURES             Location/Qualifiers
     source          1..5285
                     /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        1464..1732
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1753..1827
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1832..1851
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1953..1970)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2124..2712)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2204..2223)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2883..3674,3675..3743))
                     /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     3506..3525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3744..3848)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3875..5217
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4838..4885)
                     /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     5258..5276
                     /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 ggcgcgtcca ttcgccattc aggctgcgca
     1381 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gaatgcgtgc
     1441 gatagggaac aaaagctgga gcttctttga aaagataatg tatgattatg ctttcactca
     1501 tatttataca gaaacttgat gttttctttc gagtatatac aaggtgatta catgtacgtt
     1561 tgaagtacaa ctctagattt tgtagtgccc tcttgggcta gcggtaaagg tgcgcatttt
     1621 ttcacaccct acaatgttct gttcaaaaga ttttggtcaa acgctgtaga agtgaaagtt
     1681 ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa agataaatga tcgttgacca
     1741 gttgatcagt tggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     1801 acttgaaaaa gtggcaccga gtcggtggtg ctttttttgt tttttatgtc ttcgagtcat
     1861 gtaattagtt aatcgcgtgc attccagctg cattaatgaa tcggccaacg cgcggggaga
     1921 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1981 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     2041 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2101 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     2161 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2221 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2281 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     2341 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     2401 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2461 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2521 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     2581 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2641 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2701 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2761 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2821 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2881 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2941 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     3001 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     3061 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     3121 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     3181 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     3241 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     3301 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     3361 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     3421 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     3481 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3541 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3601 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3661 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3721 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3781 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3841 gttccgcgca catttccccg aaaagtgcca cctgaacgaa gcatctgtgc ttcattttgt
     3901 agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt
     3961 tacagaacag aaatgcaacg cgaaagcgct attttaccaa cgaagaatct gtgcttcatt
     4021 tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc
     4081 atttttacag aacagaaatg caacgcgaga gcgctatttt accaacaaag aatctatact
     4141 tcttttttgt tctacaaaaa tgcatcccga gagcgctatt tttctaacaa agcatcttag
     4201 attacttttt ttctcctttg tgcgctctat aatgcagtct cttgataact ttttgcactg
     4261 taggtccgtt aaggttagaa gaaggctact ttggtgtcta ttttctcttc cataaaaaaa
     4321 gcctgactcc acttcccgcg tttactgatt actagcgaag ctgcgggtgc attttttcaa
     4381 gataaaggca tccccgatta tattctatac cgatgtggat tgcgcatact ttgtgaacag
     4441 aaagtgatag cgttgatgat tcttcattgg tcagaaaatt atgaacggtt tcttctattt
     4501 tgtctctata tactacgtat aggaaatgtt tacattttcg tattgttttc gattcactct
     4561 atgaatagtt cttactacaa tttttttgtc taaagagtaa tactagagat aaacataaaa
     4621 aatgtagagg tcgagtttag atgcaagttc aaggagcgaa aggtggatgg gtaggttata
     4681 tagggatata gcacagagat atatagcaaa gagatacttt tgagcaatgt ttgtggaagc
     4741 ggtattcgca atattttagt agctcgttac agtccggtgc gtttttggtt ttttgaaagt
     4801 gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa gttcctatac tttctagaga
     4861 ataggaactt cggaatagga acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc
     4921 aacgcgagct gcgcacatac agctcactgt tcacgtcgca cctatatctg cgtgttgcct
     4981 gtatatatat atacatgaga agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta
     5041 tatgcgtcta tttatgtagg atgaaaggta gtctagtacc tcctgtgata ttatcccatt
     5101 ccatgcgggg tatcgtatgc ttccttcagc actacccttt agctgttcta tatgctgcca
     5161 ctcctcaatt ggattagtct catccttcaa tgctatcatt tcctttgata ttggatcata
     5221 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     5281 tcgtc
//
