pCMV-T7-SpG-P2A-EGFP (RTW4177)

LOCUS       pCMV-T7-SpG-P2A-EGFP (RTW4177)               8451 bp ds-DNA     circular SYN 19-MAR-2020
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            SpG(D1135L/S1136W/G1218K/E1219Q/R1335Q/T1337R) with a c-terminal 
            bi-partite NLS, 3x flag tag, and P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-T7-SpG-P2A-EGFP (RTW4177)
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  JOURNAL   unpublished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 19, 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"
     enhancer        100..479
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        480..683
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     634..654
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     725..744
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        725..743
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      751..760
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             757..2475
                     /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             4915..4935
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4948..5013
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5032..5088
                     /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             5089..5808
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5134..5155)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5395..5414)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5742..5763
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5814..5831
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5854..5871)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5860..6084
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6155..6171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6155..6171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6168..6190)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6179..6195
                     /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(6203..6233)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6248..6269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6386..6403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6557..7145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6637..6656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7316..8176)
                     /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     7939..7958
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8177..8281)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8360..8379)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
       61 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
      121 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      181 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      241 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      301 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      361 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      421 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      481 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
      541 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      601 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      661 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag agatccgcgg
      721 ccgctaatac gactcactat agggagagcc gccaccatgg acaagaagta cagcatcggc
      781 ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
      841 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
      901 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
      961 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     1021 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     1081 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     1141 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     1201 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     1261 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     1321 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     1381 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     1441 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg
     1501 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     1561 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     1621 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     1681 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     1741 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     1801 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
     1861 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     1921 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     1981 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     2041 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     2101 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     2161 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
     2221 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     2281 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
     2341 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
     2401 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
     2461 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
     2521 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
     2581 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
     2641 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
     2701 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac
     2761 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
     2821 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
     2881 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
     2941 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
     3001 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
     3061 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
     3121 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
     3181 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
     3241 gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag
     3301 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac
     3361 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac
     3421 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg
     3481 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
     3541 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag
     3601 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
     3661 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
     3721 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
     3781 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
     3841 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
     3901 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
     3961 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc
     4021 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
     4081 tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct
     4141 aagaagtacg gcggcttcct gtggcccacc gtggcctatt ctgtgctggt ggtggccaaa
     4201 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc
     4261 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
     4321 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
     4381 ggccggaaga gaatgctggc ctctgccaag cagctgcaga agggaaacga actggccctg
     4441 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
     4501 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag
     4561 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
     4621 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat
     4681 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
     4741 accaccatcg accggaagca gtacagaagc accaaagagg tgctggacgc caccctgatc
     4801 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac
     4861 ggatccggcg gaggcggaag cgggaaaaga accgccgacg gcagcgaatt cgagcccaag
     4921 aagaagagga aagtctcgag cggaggcgac tacaaagacc atgacggtga ttataaagat
     4981 catgacatcg attacaagga tgacgatgac aagggaggcg gtggaagcgg cgcaacaaac
     5041 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgat ggtgagcaag
     5101 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     5161 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     5221 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     5281 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     5341 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     5401 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     5461 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     5521 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     5581 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     5641 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     5701 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     5761 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtgacc ggtcatcatc
     5821 accatcacca ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     5881 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     5941 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     6001 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     6061 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgatac
     6121 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     6181 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg
     6241 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     6301 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6361 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     6421 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6481 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6541 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6601 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6661 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6721 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6781 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6841 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6901 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6961 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     7021 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     7081 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7141 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7201 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7261 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7321 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7381 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7441 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7501 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7561 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7621 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7681 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7741 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7801 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7861 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7921 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7981 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     8041 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     8101 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     8161 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     8221 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     8281 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc
     8341 ccctagggtc gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     8401 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa g
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