LOCUS       Exported                8465 bp ds-DNA     circular SYN 24-AUG-2021
DEFINITION  mammalian expression of GFP-Cas. Note-This plasmid contains GFP21, 
            which has a sequence similar to YFP, but emission/excitation similar
            to GFP..
ACCESSION   .
VERSION     .
KEYWORDS    pGFP Cas
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8465)
  AUTHORS   Yamada KM
  TITLE     Yamada Lab Plasmids
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 8465)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 24, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8465
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    33..54
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        69..99
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    107..123
                     /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)."
     primer_bind     112..134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     131..147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     131..147
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        176..555
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        556..759
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     710..730
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     756..780
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        990..1008
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     990..1007
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1021..1040
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1086..1095
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1092..1808
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1137..1158)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1398..1417)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1745..1766
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4443..4466
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      4927..4936
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     polyA_signal    4940..5074
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4977..4996)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5031..5050
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        5143..5500
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(5166..5186)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5351..5486
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5413..5432
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(5520..5537)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5520..5536)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5529..5551)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5749..6204
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5836..5855)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6046..6067
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6221..6240)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6340..6362
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6400..6418)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6486..6590
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6591..7451
                     /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     complement(6809..6828)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7622..8210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8111..8130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8364..8381
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
       61 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      121 caatttcaca caggaaacag ctatgacatg attacgaatt aattcgagct cgcccgacat
      181 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
      241 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      301 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      361 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      421 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      481 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      541 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
      601 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      661 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
      721 ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc
      781 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc
      841 ctccgcggcc gggaacggtg cattggaacg cggattcccc gtgccaagag tgacgtaagt
      901 accgcctata gagtctatag gcccaccccc ttggcttcgt tagaacgcgg ctacaattaa
      961 tacataacct tatgtatcat acacatacga tttaggtgac actatagaat aacatccact
     1021 ttgcctttct ctccacaggt gtccactccc aggtccaact gcacctcggt tctatcgatt
     1081 cagccgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1141 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1201 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1261 cctggcccac cctcgtgacc accttcgcct acggcctgca gtgcttcgcc cgctaccccg
     1321 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1381 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1441 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1501 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1561 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1621 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1681 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     1741 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1801 tgtacaaggg aggcggatcc atgaagtacc tgaacgtgct ggccaaagcc ctctatgaca
     1861 acgtagctga gtctccggat gaactctcct tccgaaaggg tgacatcatg acagtgctcg
     1921 agcgggacac tcagggcctg gatggctggt ggctctgctc actgcatggg cgccagggca
     1981 ttgtgcctgg taaccgcctc aagattctgg ttggcatgta tgacaagaag ccagcagcac
     2041 ctggaccagg cccacctgcc accccacccc agccccagcc cagccttccc cagggcgtcc
     2101 atactccagt gcctccagct tcccagtaca gtcccatgct ccccactgca taccaacccc
     2161 aacctgacaa tgtgtacctg gtacccactc ccagcaagac tcagcaaggt ctctaccaag
     2221 cccctgggcc caacccacag ttccagtcac ccccagccaa acaaacatcc acattctcaa
     2281 agcaaacacc tcatcactca tttcccagcc ctgccacaga cctttaccag gtgcctccag
     2341 ggcctggaag tccagcccag gacatttacc aggtgccacc ttccgctggc acagggcatg
     2401 acatctacca agtccctcca tctttggaca cccgcagctg ggagggaacg aagccaccgg
     2461 caaaggtggt ggtacctact cgagtgggac agggctatgt gtacgaggcc tcccaggcag
     2521 aacaggatga gtacgacacc ccgcgccacc tgctagctcc aggttcccag gacatctatg
     2581 atgtgccccc tgttcgagga ctgcttccca accagtatgg ccaggaggta tatgacaccc
     2641 cgcctatggc agtcaaaggc cccaatggcc gagacccgtt gttggatgtg tatgacgtgc
     2701 cccctagtgt ggagaaaggc ctgccgccgt ccaaccatca ttcggtgtat gatgttcctc
     2761 cttctgtgag caaggatgtg cctgatggcc cactgctgcg tgaggaaaca tatgatgtac
     2821 cccctgcctt cgccaaaccc aagccttttg atccgacccg acatccatta atcctcgctg
     2881 cacctcctcc ggactcccca ccagctgagg atgtgtatga tgtgccaccc cctgctcctg
     2941 acctctatga tgtgccccct ggtttgcggc ggcctggccc tggaacactg tatgacgtgc
     3001 ctcgagagcg ggtgctccct cctgaggtgg ctgatgggag tgtaattgat gatggtgtgt
     3061 atgctgtgcc cccaccagcc gagcgagagg cgccaacaga tggcaagcgc ctgtcggcct
     3121 ctagcacggg cagcacacgc agcagccaat cggcgtcttc cttggaggta gtggtgccag
     3181 gtcgggagcc cctggagctg gaagttgctg tggagaccct ggctcggcta cagcagggtg
     3241 tgagcaccac cgtagcccac cttctggacc tggtgggcag tgccagtggc cccgggggct
     3301 ggcgtagtac ctctgagccc caggagcctc ccgtgcagga cctgaaagct gcagtggccg
     3361 cagttcatgg ggcagtacat gagctcctgg agtttgcccg cagtgcagtg agcagtgcca
     3421 ctcacacttc tgaccgcact ttgcacgcca agcttagccg gcagctacag aagatggagg
     3481 atgtgtacca gacactggtg gtccatggtc aggtccttga cagtggccgg ggaggtccag
     3541 gattcactct ggacgacctg gaccgcctgg tggcctgctc acgggctgtg cccgaggatg
     3601 ccaagcagct ggcctccttt ttgcatggta atgcctcgct gcttttcaga cggaccaaag
     3661 cccctggccc cgggcctgag ggcagcagct ccctgcacct caaccccacc gataaagcca
     3721 gcagcatcca gtcacgccct ctaccctcac ctccaaagtt cacctcccag gactctccgg
     3781 atggccagta tgagaacagt gaagggggtt ggatggagga ctatgactac gttcatctgc
     3841 aggggaagga ggaatttgag aagacccaga aggaactgct ggaaaagggc aacatcgtgc
     3901 gacagggaaa aggccaactg gagctgcagc agctgaaaca gtttgagcga ctggagcagg
     3961 aggtgtctcg tcccatagac catgacctgg ccaactggac accagcccag ccccttgtgc
     4021 cggggcggac agggggactg gggccttcag accgacagct gctgctcttc tacttggagc
     4081 agtgtgaggc caacctgacc acactgacag atgcggtgga cgccttcttc accgcagtgg
     4141 ccaccaacca gccacccaag atctttgtgg cacacagcaa gtttgtcatt cttagtgcac
     4201 acaagcttgt gttcattggg gacacactgt cacggcaggc aaaggcagct gatgtgcgaa
     4261 gccaagtgac ccactacagc aatctgctgt gtgacctcct gcgtggcatc gtggccacca
     4321 ccaaggccgc tgccctgcag tacccatccc catctgctgc ccaggacatg gtagacaggg
     4381 tcaaggagct aggccacagc actcagcagt tccgccgtgt cctgggccag ctggctgccg
     4441 ccgactacaa ggacgacgat gacaagtgaa agcagaagac tgggctgtga ggctggggag
     4501 tgggtagcgg tggtgtgagc tacccaggcg ctcgtcttga agtggagatc ctgcaggttt
     4561 ggggacaggt aaccccagct ctgctttggg cctggtgccc tcaactgtcc agggatttgt
     4621 acatatttat atcaaggcag gatgcagaat gcctcctcgg agaggctgag gagccccgtc
     4681 agagtggacc atgggctggg atcaccaggg ttggggcaca tgggctccag tccacagggc
     4741 tccctgggac aggaagtaca gtgtggtgta cacctctgca ccaagaaaac ctaaagaact
     4801 atttttcact attgattttt ttccaatcat ttgactaata gtctacattt aataaaattt
     4861 taaaaaaaaa aaaaaaaagg atccgcggcc gccgaattct agagtcgacc tgcagaagct
     4921 agcttggccg ccatggccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4981 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5041 caaactcatc aatgtatctt atcatgtctg gatcgggaat taattcggcg cagcaccatg
     5101 gcctgaaata acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca
     5161 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     5221 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     5281 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     5341 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     5401 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     5461 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctgttaa cagcttggca
     5521 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     5581 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     5641 ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
     5701 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     5761 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     5821 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     5881 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     5941 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     6001 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     6061 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc
     6121 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     6181 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     6241 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     6301 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     6361 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     6421 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     6481 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6541 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6601 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6661 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6721 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6781 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6841 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6901 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6961 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     7021 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     7081 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     7141 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     7201 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     7261 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     7321 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     7381 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     7441 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     7501 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     7561 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7621 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7681 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7741 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     7801 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7861 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7921 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7981 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     8041 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     8101 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     8161 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     8221 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     8281 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     8341 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     8401 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     8461 ttccc
//
