LOCUS       pCMV-T7-ABE8e-SpCas9(LWKYQS)-P2A-EGFP_(AHK404) 9033 bp    DNA     circular SYN 02-OCT-2024
DEFINITION  .
ACCESSION   pCMV-T7-ABE8e-SpCas9(LWKYQS)-P2A-EGFP_(AHK404)
VERSION     pCMV-T7-ABE8e-SpCas9(LWKYQS)-P2A-EGFP_(AHK404)
KEYWORDS    pCMV_ABEmax_P2A_GFP.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
            .
REFERENCE   1  (bases 1 to 9627)
  AUTHORS   Koblan LW, Doman JL, Wilson C, Levy JM, Tay T, Newby GA, Maianti JP,
            Raguram A and Liu DR
  TITLE     Improving cytidine and adenine base editors by expression
            optimization and ancestral reconstruction.
  JOURNAL   Nat Biotechnol. 2018 May 29. pii: nbt.4172. doi: 10.1038/nbt.4172.
   PUBMED   29813047
REFERENCE   2  (bases 1 to 9627)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     regulatory      173..552
                     /note="Geneious type: enhancer"
                     /standard_name="CMV enhancer"
     regulatory      553..756
                     /note="Geneious type: promoter"
                     /standard_name="CMV promoter"
     regulatory      798..816
                     /note="Geneious type: promoter"
                     /standard_name="T7 promoter"
     misc_feature    821..833
                     /note="Geneious type: motif"
                     /standard_name="Kozak"
     CDS             830..832
                     /standard_name="start"
     CDS             833..886
                     /standard_name="BPNLS(SV40)"
     CDS             884..1384
                     /standard_name="TadA*8e"
     misc_feature    950..952
                     /standard_name="W23R"
     misc_feature    989..991
                     /standard_name="H36L"
     misc_feature    1025..1027
                     /standard_name="P48A"
     misc_feature    1034..1036
                     /standard_name="R51L"
     misc_feature    1133..1135
                     /standard_name="L84F"
     misc_feature    1199..1201
                     /standard_name="A106V"
     misc_feature    1205..1207
                     /standard_name="D108N"
     misc_feature    1208..1210
                     /standard_name="A109S"
     misc_feature    1214..1216
                     /standard_name="T111R"
     misc_feature    1238..1240
                     /standard_name="D119N"
     misc_feature    1247..1249
                     /standard_name="H122N"
     misc_feature    1250..1252
                     /standard_name="H123Y"
     misc_feature    1319..1321
                     /standard_name="S146C"
     misc_feature    1322..1324
                     /standard_name="D147 (changed back from D147Y in ABE7.10)"
     misc_feature    1328..1330
                     /standard_name="F149Y"
     misc_feature    1337..1339
                     /standard_name="R152P"
     misc_feature    1346..1348
                     /standard_name="E155V"
     misc_feature    1349..1351
                     /standard_name="I156F"
     misc_feature    1352..1354
                     /standard_name="K157N"
     misc_feature    1379..1381
                     /standard_name="T166I"
     misc_feature    1382..1384
                     /standard_name="D167N"
     CDS             1385..1480
                     /standard_name="linker"
     misc_difference 1505..1507
                     /standard_name="D10A"
     CDS             5582..5593
                     /standard_name="linker"
     CDS             5594..5644
                     /standard_name="BPNLS(SV40)"
     CDS             5645..5653
                     /standard_name="linker"
     CDS             5654..5710
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding
                     separatepolypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
                     /standard_name="P2A"
     CDS             5711..6427
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="mammalian codon-optimized"
                     /standard_name="EGFP"
     CDS             6428..6439
                     /standard_name="linker"
     CDS             6440..6460
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
                     /standard_name="NLS(SV40)"
     CDS             6461..6463
                     /standard_name=""
     misc_feature    6515..6739
                     /note="Geneious type: polyA_signal"
     rep_origin      complement(7212..7800)
     CDS             complement(7971..8831)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
                     /standard_name="AmpR"
     regulatory      complement(8832..8936)
                     /note="Geneious type: promoter"
     motif           4880..4882
                     /standard_name="L1135"
     motif           4883..4885
                     /standard_name="W1136"
     motif           5129..5131
                     /standard_name="K1218"
     motif           5132..5134
                     /standard_name="Y1219"
     motif           5480..5482
                     /standard_name="Q1335"
     motif           5486..5488
                     /standard_name="S1337"
     CDS             1478..5581
                     /product="nickase mutant of the Cas9 endonuclease from
                     theStreptococcus pyogenes Type II CRISPR/Cas system"
                     /note="generates RNA-guided single strand nicks in DNA
                     dueto the D10A mutation in the RuvC catalytic domain"
                     /standard_name="nSpCas9(D10A)-LWKYQS"
ORIGIN
        1 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
       61 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
      121 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
      181 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      241 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      301 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      361 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      421 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      481 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      541 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      601 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      661 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      721 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc
      781 tagagatccg cggccgctaa tacgactcac tatagggaga gccgccacca tgaaacggac
      841 agccgacgga agcgagttcg agtcaccaaa gaagaagcgg aaagtctctg aggtggagtt
      901 ttcccacgag tactggatga gacatgccct gaccctggcc aagagggcac gcgatgagag
      961 ggaggtgcct gtgggagccg tgctggtgct gaacaataga gtgatcggcg agggctggaa
     1021 cagagccatc ggcctgcacg acccaacagc ccatgccgaa attatggccc tgagacaggg
     1081 cggcctggtc atgcagaact acagactgat tgacgccacc ctgtacgtga cattcgagcc
     1141 ttgcgtgatg tgcgccggcg ccatgatcca ctctaggatc ggccgcgtgg tgtttggcgt
     1201 gaggaacagc aaaagaggcg ccgcaggctc cctgatgaac gtgctgaact accccggcat
     1261 gaatcaccgc gtcgaaatta ccgagggaat cctggcagat gaatgtgccg ccctgctgtg
     1321 cgatttctac cggatgccta gacaggtgtt caatgctcag aagaaggccc agagctccat
     1381 caactccgga ggatctagcg gaggctcctc tggctctgag acacctggca caagcgagag
     1441 cgcaacacct gaaagcagcg ggggcagcag cggggggtca gacaagaagt acagcatcgg
     1501 cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     1561 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     1621 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     1681 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     1741 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     1801 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     1861 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     1921 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     1981 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     2041 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     2101 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     2161 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     2221 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     2281 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     2341 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     2401 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     2461 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     2521 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     2581 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     2641 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     2701 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     2761 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     2821 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     2881 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     2941 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     3001 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     3061 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     3121 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     3181 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     3241 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     3301 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     3361 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     3421 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     3481 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     3541 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     3601 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     3661 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     3721 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     3781 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     3841 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     3901 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     3961 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
     4021 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     4081 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     4141 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     4201 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     4261 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     4321 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     4381 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     4441 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
     4501 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     4561 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     4621 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
     4681 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     4741 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     4801 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     4861 taagaagtac ggcggcttcc tgtggcccac cgtggcctat tctgtgctgg tggtggccaa
     4921 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     4981 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     5041 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     5101 cggccggaag agaatgctgg cctctgccaa gtacctgcag aagggaaacg aactggccct
     5161 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     5221 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     5281 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     5341 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     5401 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     5461 caccaccatc gaccggaagc agtacagcag caccaaagag gtgctggacg ccaccctgat
     5521 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggtga
     5581 ctctggcggc tcaaaaagaa ccgccgacgg cagcgaattc gagcccaaga agaagaggaa
     5641 agtcggaagc ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa
     5701 ccctggacct atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     5761 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     5821 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     5881 ctggcccacc ctcgtgacca ccctgaccta tggagtgcag tgcttcagcc gctaccccga
     5941 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     6001 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     6061 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     6121 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     6181 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     6241 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     6301 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     6361 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     6421 gtacaagtct ggtggttctc ccaagaagaa gaggaaagtc taaccggtca tcatcaccat
     6481 caccattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     6541 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     6601 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     6661 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     6721 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg
     6781 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     6841 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc
     6901 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6961 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     7021 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     7081 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     7141 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     7201 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     7261 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     7321 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     7381 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     7441 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     7501 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     7561 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     7621 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     7681 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     7741 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     7801 gaagatcctt tgatcttttc tacggggtct gacactcagt ggaacgaaaa ctcacgttaa
     7861 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     7921 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     7981 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     8041 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     8101 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     8161 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     8221 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     8281 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     8341 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     8401 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     8461 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     8521 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     8581 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     8641 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     8701 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     8761 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     8821 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     8881 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     8941 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta
     9001 gggtcgactc tcagtacaat ctgctctgat gcc
//
