LOCUS       40924_450        7691 bp DNA     circular SYN 13-MAY-2021
DEFINITION  AAV genome: expresses the N-terminal of v5 AAV-CBE from the Cbh 
            promoter, and U6-sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7691)
  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 7691)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7691)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41551-019-0501-5"; journalName: "Nat Biomed Eng"; date: 
            "2020-01"; volume: "4"; issue: "1"; pages: "97-110"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(125..141)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      283..738
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        764..868
                     /label=AmpR promoter
     CDS             869..1726
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1900..2488
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2642..2659
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2776..2797
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2812..2842
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2850..2866
                     /label=lac operator
                     /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     2874..2890
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2917..3046
                     /label=AAV2 ITR
     enhancer        3069..3354
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        3356..3633
                     /label=chicken beta-actin promoter
     intron          3634..3861
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3915..3935
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             3933..4619
                     /codon_start=1
                     /label=APOBEC-1
                     /note="cytidine deaminase (C to U editing enzyme) from rat"
                     /translation="VSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             4716..6431
                     /codon_start=1
                     /label=Cas9(N)
                     /note="N-terminal portion of Streptococcus pyogenes Cas9 
                     (Zetsche et al., 2015)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             6780..6800
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     misc_feature    6810..7057
                     /label=WPRE3
                     /note="Truncated short version of woodchuck hepatitis virus
                     posttranscriptional regulatory element (248 bp)"
     polyA_signal    7061..7285
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(7299..7374)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7404..7644)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
ORIGIN
        1 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
       61 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta
      121 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
      181 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
      241 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
      301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
      541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
      601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
      661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
      721 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      781 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      841 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      901 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      961 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1021 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1081 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1141 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1201 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1261 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1321 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1381 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1441 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1501 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1561 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1621 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1681 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1741 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1801 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1861 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1921 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1981 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2041 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2101 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2161 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2221 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2281 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2341 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2401 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2461 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2521 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2581 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2641 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     2701 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2761 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     2821 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     2881 cagctatgac catgattacg ccagatttaa ttaaggctgc gcgctcgctc gctcactgag
     2941 gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     3001 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagatc
     3061 agggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     3121 cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca atgggtggag
     3181 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     3241 cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta catgacctta
     3301 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag
     3361 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg
     3421 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc
     3481 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
     3541 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     3601 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
     3661 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
     3721 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg
     3781 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg
     3841 cctgaaatca ctttttttca ggttggaccg gtgccaccat gaaacggaca gccgacggaa
     3901 gcgagttcga gtcaccaaag aagaagcgga aagtctcctc agagactggg cctgtcgccg
     3961 tcgatccaac cctgcgccgc cggattgaac ctcacgagtt tgaagtgttc tttgaccccc
     4021 gggagctgag aaaggagaca tgcctgctgt acgagatcaa ctggggaggc aggcactcca
     4081 tctggaggca cacctctcag aacacaaata agcacgtgga ggtgaacttc atcgagaagt
     4141 ttaccacaga gcggtacttc tgccccaata ccagatgtag catcacatgg tttctgagct
     4201 ggtccccttg cggagagtgt agcagggcca tcaccgagtt cctgtccaga tatccacacg
     4261 tgacactgtt tatctacatc gccaggctgt atcaccacgc agacccaagg aataggcagg
     4321 gcctgcgcga tctgatcagc tccggcgtga ccatccagat catgacagag caggagtccg
     4381 gctactgctg gcggaacttc gtgaattatt ctcctagcaa cgaggcccac tggcctaggt
     4441 acccacacct gtgggtgcgc ctgtacgtgc tggagctgta ttgcatcatc ctgggcctgc
     4501 ccccttgtct gaatatcctg cggagaaagc agccccagct gaccttcttt acaatcgccc
     4561 tgcagtcttg tcactatcag aggctgccac cccacatcct gtgggccaca ggcctgaagt
     4621 ctggaggatc tagcggagga tcctctggca gcgagacacc aggaacaagc gagtcagcaa
     4681 caccagagag cagtggcggc agcagcggcg gcagcgacaa gaagtacagc atcggcctgg
     4741 ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
     4801 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
     4861 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
     4921 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
     4981 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
     5041 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
     5101 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     5161 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     5221 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
     5281 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     5341 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     5401 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc
     5461 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     5521 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     5581 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     5641 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     5701 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     5761 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
     5821 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     5881 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     5941 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     6001 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     6061 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     6121 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
     6181 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     6241 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
     6301 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
     6361 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     6421 agaaaatcga gtgcctgtcc tacgagacag agatcctgac agtggagtat ggcctgctgc
     6481 caatcggcaa gatcgtggag aagaggatcg agtgtaccgt gtactctgtg gataacaatg
     6541 gcaacatcta tacacagccc gtggcacagt ggcacgatag gggagagcag gaggtgttcg
     6601 agtattgcct ggaggacggc agcctgatca gggcaaccaa ggaccacaag ttcatgacag
     6661 tggatggcca gatgctgccc atcgacgaga ttttcgagcg ggagctggac ctgatgagag
     6721 tggataacct gcctaatagc ggaggcagta aaagaacagc agacgggagt gagtttgagc
     6781 ccaagaaaaa gagaaaggtg taagatctga taatcaacct ctggattaca aaatttgtga
     6841 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     6901 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     6961 atcctggtta gttcttgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     7021 aggggctcgg ctgttgggca ctgacaattc cgtggtgcga ctgtgccttc tagttgccag
     7081 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     7141 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     7201 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     7261 gctggggatg cggtgggctc tatggctcga gaaaaaaagc accgactcgg tgccactttt
     7321 tcaagttgat aacggactag ccttatttta acttgctatt tctagctcta aaaccgagac
     7381 gcattaatcg tctcgggtgt ttcgtccttt ccacaagata tataaagcca agaaatcgaa
     7441 atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc
     7501 aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt
     7561 tacagtcaaa ttaattctaa ttatctctct aacagccttg tatcgtatat gcaaatatga
     7621 aggaatcatg ggaaataggc cctcttcctg cccgaccttg cggccgcagg aacccctagt
     7681 gatggagttg g
//
