pCMV-T7-SpRY-P2A-EGFP (RTW4830)

LOCUS       pCMV-T7-SpRY-P2A-EGFP (RTW4830)                8451 bp ds-DNA     circular SYN 20-MAR-2020
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            SpRY(A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/R1333P/R1
            335Q/T1337R) with a c-terminal bi-partite NLS, 3x flag tag, and 
            P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-T7-SpRY-P2A-EGFP (RTW4830)
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  JOURNAL   unpublished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             complement(250..1110)
                     /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     873..892
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1111..1215)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1294..1313)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1485..1864
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1865..2068
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2019..2039
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2110..2129
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2110..2128
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2136..2145
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2142..3860
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             6300..6320
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6333..6398
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6417..6473
                     /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             6474..7193
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6519..6540)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6780..6799)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7127..7148
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7199..7216
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7239..7256)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7245..7469
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7540..7556)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7540..7556)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7553..7575)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7564..7580
                     /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(7588..7618)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7633..7654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7771..7788)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7942..79)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8022..8041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
       61 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
      121 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      181 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
      241 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
      301 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
      361 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
      421 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
      481 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
      541 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      601 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      661 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      721 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      781 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      841 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      901 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      961 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     1021 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1081 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     1141 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1201 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     1261 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
     1321 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     1381 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     1441 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     1501 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     1561 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     1621 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     1681 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     1741 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     1801 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     1861 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     1921 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     1981 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     2041 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc
     2101 cgcggccgct aatacgactc actataggga gagccgccac catggacaag aagtacagca
     2161 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     2221 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     2281 tgatcggagc cctgctgttc gacagcggcg aaacagccga gagaacccgg ctgaagagaa
     2341 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     2401 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     2461 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     2521 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     2581 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     2641 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     2701 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     2761 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     2821 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     2881 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     2941 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     3001 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     3061 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     3121 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     3181 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     3241 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     3301 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     3361 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     3421 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     3481 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     3541 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     3601 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     3661 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     3721 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     3781 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     3841 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     3901 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     3961 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     4021 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     4081 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     4141 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     4201 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     4261 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     4321 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     4381 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     4441 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     4501 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     4561 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     4621 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     4681 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     4741 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     4801 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     4861 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     4921 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     4981 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     5041 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     5101 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     5161 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     5221 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     5281 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     5341 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     5401 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     5461 aagagtctat cagacccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     5521 accctaagaa gtacggcggc ttcctgtggc ccaccgtggc ctattctgtg ctggtggtgg
     5581 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     5641 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     5701 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     5761 aaaacggccg gaagagaatg ctggcctctg ccaagcagct gcagaaggga aacgaactgg
     5821 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     5881 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     5941 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     6001 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     6061 agaatatcat ccacctgttt accctgacca gactgggagc ccctagagcc ttcaagtact
     6121 ttgacaccac catcgacccc aagcagtaca gaagcaccaa agaggtgctg gacgccaccc
     6181 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     6241 gcgacggatc cggcggaggc ggaagcggga aaagaaccgc cgacggcagc gaattcgagc
     6301 ccaagaagaa gaggaaagtc tcgagcggag gcgactacaa agaccatgac ggtgattata
     6361 aagatcatga catcgattac aaggatgacg atgacaaggg aggcggtgga agcggcgcaa
     6421 caaacttctc tctgctgaaa caagccggag atgtcgaaga gaatcctgga ccgatggtga
     6481 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     6541 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     6601 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     6661 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     6721 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     6781 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     6841 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     6901 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     6961 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     7021 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     7081 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     7141 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tgaccggtca
     7201 tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     7261 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     7321 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     7381 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     7441 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     7501 gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     7561 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     7621 ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     7681 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     7741 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     7801 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     7861 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     7921 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     7981 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     8041 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     8101 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     8161 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     8221 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     8281 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     8341 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     8401 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat c
//