pCMV-T7-SpG-HF1-P2A-EGFP (RTW5000)

LOCUS       pCMV-T7-SpG-HF1-P2A-EGFP  (RTW5000)                8451 bp ds-DNA     circular SYN 20-MAR-2020
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            SpG-HF1(SpG=D1135L/S1136W/G1218K/E1219Q/R1335Q/T1337R;HF1=N497A/R661
            A/Q695A/Q926A) with a c-terminal bi-partite NLS, 3x flag tag, and 
            P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-T7-SpG-HF1-P2A-EGFP  (RTW5000)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8451)
  AUTHORS   Kleinstiver
  TITLE     Kleinstiver lab CRISPR plasmids
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 8451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 20, 2020 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8451
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             4036..4056
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4069..4134
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4153..4209
                     /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             4210..4929
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4255..4276)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4516..4535)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4863..4884
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4935..4952
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4975..4992)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4981..5205
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5276..5292)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5276..5292)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5289..5311)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5300..5316
                     /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(5324..5354)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5369..5390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5507..5524)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5678..6266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5758..5777)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6437..7297)
                     /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     7060..7079
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7298..7402)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7481..7500)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7672..8051
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8052..8255
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8206..8226
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     8297..8316
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8297..8315
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      8323..8332
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             8329..1596
                     /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
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
ORIGIN
        1 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
       61 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
      121 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
      181 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
      241 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
      301 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
      361 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
      421 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
      481 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
      541 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
      601 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
      661 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
      721 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
      781 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
      841 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
      901 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
      961 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     1021 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     1081 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     1141 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     1201 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     1261 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     1321 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccgcctt cgataagaac
     1381 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     1441 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     1501 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     1561 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     1621 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     1681 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     1741 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     1801 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcgcc
     1861 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     1921 ttcctgaagt ccgacggctt cgccaacaga aacttcatgg ccctgatcca cgacgacagc
     1981 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     2041 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     2101 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     2161 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     2221 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     2281 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     2341 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     2401 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     2461 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     2521 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     2581 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     2641 gtggaaaccc gggccatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     2701 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     2761 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     2821 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     2881 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     2941 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     3001 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     3061 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     3121 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     3181 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     3241 agaaagaagg actgggaccc taagaagtac ggcggcttcc tgtggcccac cgtggcctat
     3301 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     3361 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     3421 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     3481 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccaa gcagctgcag
     3541 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     3601 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     3661 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     3721 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     3781 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     3841 gccgccttca agtactttga caccaccatc gaccggaagc agtacagaag caccaaagag
     3901 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     3961 ctgtctcagc tgggaggcga cggatccggc ggaggcggaa gcgggaaaag aaccgccgac
     4021 ggcagcgaat tcgagcccaa gaagaagagg aaagtctcga gcggaggcga ctacaaagac
     4081 catgacggtg attataaaga tcatgacatc gattacaagg atgacgatga caagggaggc
     4141 ggtggaagcg gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt cgaagagaat
     4201 cctggaccga tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     4261 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     4321 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     4381 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     4441 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     4501 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     4561 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     4621 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     4681 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     4741 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     4801 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     4861 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     4921 tacaagtgac cggtcatcat caccatcacc attgagttta aacccgctga tcagcctcga
     4981 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     5041 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     5101 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     5161 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     5221 gaaccagctg gggctcgata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     5281 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5341 gcataaagtg taaagcctag ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5401 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5461 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5521 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5581 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5641 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5701 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5761 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5821 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5881 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5941 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6001 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6061 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6121 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6181 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6241 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6301 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6361 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6421 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6481 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6541 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6601 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6661 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6721 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6781 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6841 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6901 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6961 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7021 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7081 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7141 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7201 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7261 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7321 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7381 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     7441 cgggagatcg atctcccgat cccctagggt cgactctcag tacaatctgc tctgatgccg
     7501 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     7561 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     7621 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
     7681 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     7741 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     7801 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     7861 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     7921 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     7981 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     8041 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     8101 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     8161 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     8221 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
     8281 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggacaagaag
     8341 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     8401 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca c
//