pCMV-T7-SpCas9-NG-P2A-EGFP (RTW3677)

LOCUS       pCMV-T7-SpCas9-NG-P2A-EGFP (RTW3677)                8451 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            SpCas9-NG(L1111R/D1135V/G1218R/E1219F/A1322R/R1335V/T1337R) with a 
            c-terminal bi-partite NLS, 3x flag tag, and P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-T7-SpCas9-NG-P2A-EGFP (RTW3677)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8451)
  AUTHORS   Walton RT, Christie KA, Whittaker MN, Kleinstiver BP
  TITLE     Unconstrained genome targeting with near-PAMless engineered 
            CRISPR-Cas9 variants.
  JOURNAL   Science. 2020 Apr 17;368(6488):290-296. doi: 
            10.1126/science.aba8853. Epub 2020 Mar 26.
  PUBMED    32217751
REFERENCE   2  (bases 1 to 8451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8451
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        134..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        514..717
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     668..688
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     759..778
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        759..777
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      785..794
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             791..2509
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             4949..4969
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4982..5047
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5066..5122
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5123..5839
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5168..5189)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5429..5448)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5776..5797
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5848..5865
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5888..5905)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5894..6118
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6189..6205)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6189..6205)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6202..6224)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6213..6229
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(6237..6267)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6282..6303
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6420..6437)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6591..7179)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6671..6690)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7350..8210)
                     /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     7973..7992
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8211..8315)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8394..8413)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
       61 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
      121 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      541 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      601 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      661 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
      721 ttagtgaacc gtcagatccg ctagagatcc gcggccgcta atacgactca ctatagggag
      781 agccgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg
      841 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
      901 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
      961 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     1021 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     1081 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     1141 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     1201 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     1261 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     1321 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     1381 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     1441 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     1501 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     1561 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     1621 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     1681 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     1741 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     1801 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     1861 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     1921 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     1981 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     2041 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     2101 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     2161 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     2221 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     2281 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     2341 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     2401 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     2461 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     2521 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     2581 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     2641 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     2701 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     2761 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     2821 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     2881 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     2941 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     3001 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     3061 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     3121 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     3181 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     3241 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     3301 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     3361 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     3421 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     3481 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     3541 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     3601 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     3661 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     3721 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     3781 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     3841 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     3901 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     3961 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     4021 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     4081 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc agacccaaga ggaacagcga
     4141 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgtgagccc
     4201 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     4261 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     4321 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     4381 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     4441 cagattcctg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     4501 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     4561 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     4621 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     4681 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     4741 tctgggagcc cctagagcct tcaagtactt tgacaccacc atcgaccgga aggtgtacag
     4801 aagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     4861 gacacggatc gacctgtctc agctgggagg cgacggatcc ggcggaggcg gaagcgggaa
     4921 aagaaccgcc gacggcagcg aattcgagcc caagaagaag aggaaagtct cgagcggagg
     4981 cgactacaaa gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga
     5041 tgacaaggga ggcggtggaa gcggcgcaac aaacttctct ctgctgaaac aagccggaga
     5101 tgtcgaagag aatcctggac cgatggtgag caagggcgag gagctgttca ccggggtggt
     5161 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     5221 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     5281 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     5341 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     5401 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     5461 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     5521 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     5581 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     5641 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     5701 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     5761 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     5821 catggacgag ctgtacaagt gaccggtcat catcaccatc accattgagt ttaaacccgc
     5881 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     5941 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     6001 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     6061 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     6121 tctgaggcgg aaagaaccag ctggggctcg ataccgtcga cctctagcta gagcttggcg
     6181 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     6241 atacgagccg gaagcataaa gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca
     6301 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6361 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6421 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6481 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6541 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6601 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6661 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6721 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6781 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6841 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6901 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6961 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     7021 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     7081 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     7141 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     7201 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     7261 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     7321 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7381 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7441 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7501 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7561 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7621 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7681 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7741 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7801 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7861 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7921 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7981 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     8041 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     8101 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     8161 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     8221 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     8281 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     8341 gacgtcgacg gatcgggaga tcgatctccc gatcccctag ggtcgactct cagtacaatc
     8401 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt g
//