LOCUS       sequence-411201-        7263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpG cas9C by the constitutive CASI promoter and sgRNA 
            scaffold by U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7263)
  AUTHORS   Yang L, Liu Z, Sun J, Chen Z, Gao F, Guo Y
  TITLE     Adenine base editor-based correction of the cardiac pathogenic Lmna 
            c.1621C > T mutation in murine hearts.
  JOURNAL   J Cell Mol Med. 2024 Feb;28(4):e18145. doi: 10.1111/jcmm.18145.
  PUBMED    38332517
REFERENCE   2  (bases 1 to 7263)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell Mol 
            Med."; date: "2024-02"; volume: "28(4):e18145. doi"; pages: " 
            10.1111/jcmm.18145"
COMMENT     Sequence Label: pAAV-CASI-inteinC-aa713 SpG C-U6- sgRNA scaffold
FEATURES             Location/Qualifiers
     source          1..7263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   12..141
                     /label=AAV2 ITR
     enhancer        217..520
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        522..799
                     /label=chicken beta-actin promoter
     regulatory      1273..1282
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1277..1286
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3442..3462
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3477..3532
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3531..3550)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     misc_RNA        complement(3637..3712)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3741..3989)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3799..3818)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3969..3989)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4324..4453
                     /label=AAV2 ITR
     primer_bind     complement(4472..4489)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4472..4488)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4481..4503)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4630..5085
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4717..4736)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4927..4948
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5111..5215
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5216..5284,5285..6076)
                     /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(5434..5453)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6247..6835
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6736..6755
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7123..7144
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7159..7164,7165..7182,7183..7189)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7197..7213
                     /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     7202..7224
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7221..7237
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7221..7237
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ccttaattag gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacgt
      181 agccatgctc taggaagatc ggaattcgga gttccgcgtt acataactta cggtaaatgg
      241 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      301 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      361 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      421 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      481 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt
      541 ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt
      601 ttaattattt tgtgcagcga tgggggcggg gggggggggg gggcgcgcgc sagggggggg
      661 gggggggggs gagggggggg gsggggsgag gcggagaggt gcggcggcag ccaatcagag
      721 cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
      781 gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
      841 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactaaaac aggtaagtcc
      901 ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg
      961 ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag
     1021 cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag
     1081 gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg
     1141 aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat
     1201 gatgcctcta ctaaccatgt tcatgttttc tttttttttc tacaggtcct gggtgacgaa
     1261 caggctagcg ccgccaccat ggcggcggcg tgcccggaac tgcgtcagct ggcgcagagc
     1321 gatgtgtatt gggatccgat tgtgagcatt gaaccggatg gcgtggaaga agtgtttgat
     1381 ctgaccgtgc cgggcccgca taactttgtg gcgaacgata ttattgcgca taactctggc
     1441 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     1501 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     1561 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     1621 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     1681 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     1741 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     1801 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     1861 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     1921 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     1981 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     2041 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     2101 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     2161 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     2221 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     2281 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     2341 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     2401 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     2461 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     2521 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     2581 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
     2641 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcctg
     2701 tggcccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     2761 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     2821 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     2881 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     2941 tctgccaagc agctgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     3001 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     3061 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     3121 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     3181 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     3241 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagcag
     3301 tacagaagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     3361 tacgagacac ggatcgacct gtctcagctg ggaggtgact ctggcggctc aaaaagaacc
     3421 gccgacggca gcgaattcga gcccaagaag aagaggaaag tctaaaccgg ttggctaata
     3481 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     3541 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     3601 aacatatgcc catatgaggc cctcacgcgt aaaaaagcac cgactcggtg ccactttttc
     3661 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa acctgagcag
     3721 gtggaattcc acctgcagtc ggtgtttcgt cctttccaca agatatataa agccaagaaa
     3781 tcgaaatact ttcaagttac ggtaagcata tgatagtcca ttttaaaaca taattttaaa
     3841 actgcaaact acccaagaaa ttattacttt ctacgtcacg tattttgtac taatatcttt
     3901 gtgtttacag tcaaattaat tccaattatc tctctaacag ccttgtatcg tatatgcaaa
     3961 tatgaaggaa tcatgggaaa taggccctcg gtaccggcga tgctggctgc catgaacaaa
     4021 ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc
     4081 catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt
     4141 tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc
     4201 tgactactcc cagtcatagc tgtccctctt ctcttatgga gatcggatcc gaattcccga
     4261 taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta atcattaact
     4321 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4381 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4441 agcgagcgcg cagccttaat taacctaatt cactggccgt cgttttacaa cgtcgtgact
     4501 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     4561 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     4621 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4681 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4741 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4801 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4861 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4921 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     4981 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5041 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     5101 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5161 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5221 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5281 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5341 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5401 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5461 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5521 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5581 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5641 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5701 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5761 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5821 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5881 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     5941 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6001 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6061 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6121 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6181 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6241 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6301 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6361 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6421 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6481 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6541 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6601 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6661 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6721 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6781 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6841 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6901 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     6961 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     7021 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     7081 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     7141 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     7201 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca gatttaatta
     7261 agg
//
