LOCUS       Exported                6732 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses the N-terminal split-intein fragment of ABEmax from the 
            CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    CMV_Npu-ABEmax N-terminal
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6732)
  AUTHORS   Levy JM, Yeh WH, Pendse N, Davis JR, Hennessey E, Butcher R, Koblan 
            LW, Comander J, Liu Q, Liu DR
  TITLE     Cytosine and adenine base editing of the brain, liver, retina, heart
            and skeletal muscle of mice via adeno-associated viruses.
  JOURNAL   Nat Biomed Eng. 2020 Jan;4(1):97-110. doi: 
            10.1038/s41551-019-0501-5. Epub 2020 Jan 14.
  PUBMED    31937940
REFERENCE   2  (bases 1 to 6732)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6732
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1654..3369
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             3718..3738
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3747..3764
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3787..3804)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3793..4017
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4088..4104)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4088..4104)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4101..4123)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4112..4128
                     /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(4136..4166)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4181..4202
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4319..4336)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4490..5078)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4570..4589)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5249..6109)
                     /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     5872..5891
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6110..6214)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6293..6312)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6484..131
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga agtcgagttt
      481 agccacgagt attggatgag gcacgcactg accctggcaa agcgagcatg ggatgaaaga
      541 gaagtccccg tgggcgccgt gctggtgcac aacaatagag tgatcggaga gggatggaac
      601 aggccaatcg gccgccacga ccctaccgca cacgcagaga tcatggcact gaggcaggga
      661 ggcctggtca tgcagaatta ccgcctgatc gatgccaccc tgtatgtgac actggagcca
      721 tgcgtgatgt gcgcaggagc aatgatccac agcaggatcg gaagagtggt gttcggagca
      781 cgggacgcca agaccggcgc agcaggctcc ctgatggatg tgctgcacca ccccggcatg
      841 aaccaccggg tggagatcac agagggaatc ctggcagacg agtgcgccgc cctgctgagc
      901 gatttcttta gaatgcggag acaggagatc aaggcccaga agaaggcaca gagctccacc
      961 gactctggag gatctagcgg aggatcctct ggaagcgaga caccaggcac aagcgagtcc
     1021 gccacaccag agagctccgg cggctcctcc ggaggatcct ctgaggtgga gttttcccac
     1081 gagtactgga tgagacatgc cctgaccctg gccaagaggg cacgcgatga gagggaggtg
     1141 cctgtgggag ccgtgctggt gctgaacaat agagtgatcg gcgagggctg gaacagagcc
     1201 atcggcctgc acgacccaac agcccatgcc gaaattatgg ccctgagaca gggcggcctg
     1261 gtcatgcaga actacagact gattgacgcc accctgtacg tgacattcga gccttgcgtg
     1321 atgtgcgccg gcgccatgat ccactctagg atcggccgcg tggtgtttgg cgtgaggaac
     1381 gcaaaaaccg gcgccgcagg ctccctgatg gacgtgctgc actaccccgg catgaatcac
     1441 cgcgtcgaaa ttaccgaggg aatcctggca gatgaatgtg ccgccctgct gtgctatttc
     1501 tttcggatgc ctagacaggt gttcaatgct cagaagaagg cccagagctc caccgactcc
     1561 ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca
     1621 cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc
     1681 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     1741 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     1801 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     1861 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     1921 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     1981 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     2041 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     2101 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     2161 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     2221 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     2281 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     2341 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     2401 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     2461 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     2521 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     2581 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     2641 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     2701 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     2761 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     2821 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     2881 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     2941 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     3001 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     3061 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     3121 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     3181 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     3241 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     3301 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     3361 aaaatcgagt gcctgtccta cgagacagag atcctgacag tggagtatgg cctgctgcca
     3421 atcggcaaga tcgtggagaa gaggatcgag tgtaccgtgt actctgtgga taacaatggc
     3481 aacatctata cacagcccgt ggcacagtgg cacgataggg gagagcagga ggtgttcgag
     3541 tattgcctgg aggacggcag cctgatcagg gcaaccaagg accacaagtt catgacagtg
     3601 gatggccaga tgctgcccat cgacgagatt ttcgagcggg agctggacct gatgagagtg
     3661 gataacctgc ctaatagcgg aggcagtaaa agaacagcag acgggagtga gtttgagccc
     3721 aagaaaaaga gaaaggtgta accggtcatc atcaccatca ccattgagtt taaacccgct
     3781 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     3841 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3901 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3961 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     4021 ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt
     4081 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4141 tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat
     4201 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4261 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4321 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4381 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4441 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4501 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4561 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4621 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4681 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4741 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4801 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4861 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4921 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4981 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5041 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5101 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5161 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5221 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5281 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5341 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5401 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5461 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5521 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5581 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5641 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5701 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5761 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5821 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5881 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5941 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6001 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6061 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6121 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6181 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6241 acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct
     6301 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     6361 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     6421 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     6481 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     6541 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     6601 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     6661 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     6721 atcaagtgta tc
//
