LOCUS       Exported                6824 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Express a pegRNA targeting SERPINA1, and a nicking sgRNA and the 
            N-terminal split-intein fragment of PE2.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-U6-pegSERPINA1-U6-Nicking-PE2-N
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6824)
  TITLE     Improved prime editors enable pathogenic allele correction and 
            cancer modelling in adult mice
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6824)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6824
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_RNA        140..215
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(283..299)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        330..570
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        600..675
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      1086..1095
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1101..1127
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             1128..2843
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     primer_bind     complement(3597..3614)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3603..3810
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3819..3948
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      4023..4478
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4110..4129)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4320..4341
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4495..4514)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4614..4636
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4674..4692)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4760..4864
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4865..5725
                     /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(5083..5102)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5896..6484
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6385..6404
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6546..6675
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        6694..110
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
ORIGIN
        1 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
       61 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgt
      121 cccctccagg ccgtgcatag ttttagagct agaaatagca agttaaaata aggctagtcc
      181 gttatcaact tgaaaaagtg ggaccgagtc ggtcctctcg tcgatggtca gcacagcttt
      241 atgcacggcc tggagttttt ttgaattcga tatcaagctt atcgataccg tcgacctcgg
      301 ctgccgctgg aggtgctcaa agagatggag agggcctatt tcccatgatt ccttcatatt
      361 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
      421 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
      481 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
      541 tcttggcttt atatatcttg tggaaaggac gaaacaccgt tcaatcatta agaagacaag
      601 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
      661 gcaccgagtc ggtgcttttt ttgaattcga tatcaagctt atcgataccg tcgctagccc
      721 gggtcgacta gtgatcagtg tgcgggagtg taaagctggt ttaaacgttg ttgtttgggg
      781 ttgaattact cttctagacc gcggcgcgcc tccatggata tcaagcttat ggaggcggta
      841 ctatgtagat gagaattcag gagcaaactg ggaaaagcaa ctgcttccaa atatttgtga
      901 tttttacagt gtagttttgg aaaaactctt agcctaccaa ttcttctaag tgttttaaaa
      961 tgtgggagcc agtacacatg aagttataga gtgttttaat gaggcttaaa tatttaccgt
     1021 aactatgaaa tgctacgcat atcatgctgt tcaggctccg tggccacgca actcatacta
     1081 ccggtgccac catggctagc cccgccgcca agcgcgtgaa gctggacgac aagaagtaca
     1141 gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
     1201 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
     1261 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
     1321 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     1381 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     1441 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     1501 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     1561 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     1621 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     1681 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     1741 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     1801 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc
     1861 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     1921 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     1981 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     2041 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     2101 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     2161 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg
     2221 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     2281 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     2341 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     2401 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     2461 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     2521 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     2581 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     2641 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca
     2701 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     2761 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     2821 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     2881 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     2941 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     3001 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     3061 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     3121 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     3181 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     3241 aggacatcca gaaagcccag gtgtgcctgt cctacgagac agagatcctg acagtggagt
     3301 atggcctgct gccaatcggc aagatcgtgg agaagaggat cgagtgtacc gtgtactctg
     3361 tggataacaa tggcaacatc tatacacagc ccgtggcaca gtggcacgat aggggagagc
     3421 aggaggtgtt cgagtattgc ctggaggacg gcagcctgat cagggcaacc aaggaccaca
     3481 agttcatgac agtggatggc cagatgctgc ccatcgacga gattttcgag cgggagctgg
     3541 acctgatgag agtggataac ctgcctaatt gagaattcct agagctcgct gatcagcctc
     3601 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3661 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3721 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3781 ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt
     3841 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     3901 acgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     3961 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     4021 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4081 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4141 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4201 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     4261 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4321 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     4381 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     4441 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     4501 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     4561 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4621 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     4681 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     4741 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4801 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4861 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4921 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4981 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5041 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5101 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5161 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5221 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5281 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5341 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5401 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5461 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5521 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5581 aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     5641 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     5701 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     5761 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5821 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5881 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5941 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6001 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6061 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6121 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6181 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6241 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6301 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6361 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6421 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6481 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6541 catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga
     6601 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
     6661 tcactagggg ttcctgcggc ctctagaggt accgagggcc tatttcccat gattccttca
     6721 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
     6781 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatt
//
