LOCUS       sequence-166364-        5289 bp    DNA     circular SYN 15-APR-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 5289)
  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 5289)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pCfB3042(gRNA X-4)
FEATURES             Location/Qualifiers
     source          1..5289
                     /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        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"
     primer_bind     complement(1957..1974)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2128..2716)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2208..2227)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2887..3678,3679..3747))
                     /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     3510..3529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3748..3852)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3879..5221
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4842..4889)
                     /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     5262..5280
                     /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 tccattcgcc 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 atgatccgcc
     1741 attcaagagc agcaacgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     1801 atcaacttga aaaagtggca ccgagtcggt ggtgcttttt ttgtttttta tgtcttcgag
     1861 tcatgtaatt agttaatcgc gtgcattcca gctgcattaa tgaatcggcc aacgcgcggg
     1921 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     1981 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2041 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2101 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2161 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2221 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2281 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2341 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2401 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2461 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2521 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     2581 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2641 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     2701 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     2761 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     2821 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     2881 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     2941 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3001 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3061 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3121 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3181 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3241 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3301 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3361 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3421 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3481 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3541 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3601 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3661 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3721 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     3781 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3841 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt
     3901 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca
     3961 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt
     4021 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag
     4081 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta
     4141 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc
     4201 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc
     4261 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa
     4321 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt
     4381 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga
     4441 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct
     4501 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca
     4561 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat
     4621 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt
     4681 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg
     4741 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga
     4801 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta
     4861 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa
     4921 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt
     4981 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta
     5041 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc
     5101 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct
     5161 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat
     5221 catactaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5281 cctttcgtc
//
