LOCUS       sequence-409788-        5952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cre recombinase specifically in cardiomyocytes and uses U6
            promoter to express sgRNAs targeting murine Lmna exon 10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5952)
  AUTHORS   Sun Y, Guo C, Chen Z, Lin J, Yang L, Zhang Y, Wu C, Zhao D, Jardin 
            B, Pu WT, Zhao M, Dong E, Hu X, Zhang S, Guo Y
  TITLE     Non-Cell-Autonomous Cardiomyocyte Regulation Complicates Gene 
            Supplementation Therapy for Lmna-Associated Cardiac Defects in Mice.
  JOURNAL   JACC Basic Transl Sci. 2024 Aug 21;9(11):1308-1325. doi: 
            10.1016/j.jacbts.2024.06.004. eCollection 2024 Nov.
  PUBMED    39619139
REFERENCE   2  (bases 1 to 5952)
  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"; doi: 
            "10.1016/j.jacbts.2024.06.004"; journalName: "JACC Basic Transl 
            Sci"; date: "2024-08-21- 21"; volume: "9"; issue: "11"; pages: 
            "1308-1325"
COMMENT     Sequence Label: pAAV-Tnnt2-Cre-U6-Lmna-sgRNA
FEATURES             Location/Qualifiers
     source          1..5952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     125..146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(300..319)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     419..441
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(479..497)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        565..669
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(670..738,739..1530)
                     /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(888..907)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1701..2289
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2190..2209
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2351..2480
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2502..2750
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2771..2846
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2859..3107
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3128..3203
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     intron          3755..3887
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3870..3889
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        3932..3950
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3962..3982
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5049..5525
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5248..5267)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5565..5705
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5565..5694
                     /label=AAV2 ITR
     rep_origin      join(5780..5952,1..283)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5867..5886)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
       61 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      121 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
      181 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
      241 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
      301 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
      361 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
      421 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
      481 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
      541 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
      601 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
      661 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
      721 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
      781 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
      841 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
      901 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
      961 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1021 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1081 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1141 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     1201 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     1261 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     1321 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     1381 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     1441 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     1501 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     1561 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     1621 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     1681 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     1741 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     1801 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     1861 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     1921 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     1981 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2041 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2101 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2161 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2221 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2281 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2341 gctcacatgt cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg
     2401 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa
     2461 ctccatcact aggggttcct gcggccgcac gcgccggtac cgagggccta tttcccatga
     2521 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     2581 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     2641 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     2701 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc tgagcgcacc
     2761 agcttgcgca gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     2821 ttgaaaaagt ggcaccgagt cggtgctttt ttacgcgtga gggcctattt cccatgattc
     2881 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     2941 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     3001 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     3061 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccatc ctcattgtcc
     3121 tcaaccagtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     3181 aaaaagtggc accgagtcgg tgcttttttt ctagtgggat aaaagcagtc tgggctttca
     3241 catgacagca tctggggctg cggcagaggg tcgggtccga agcgctgcct tatcagcgtc
     3301 cccagccctg ggaggtgaca gctggctggc ttgtgtcagc ccctcgggca ctcacgtatc
     3361 tccgtccgac gggtttaaaa tagcaaaact ctgaggccac acaatagctt gggcttatat
     3421 gggctcctgt gggggaaggg ggagcacgga gggggccggg gccgctgctg ccaaaatagc
     3481 agctcacaag tgttgcattc ctctctgggc gccgggcaca ttcctgctgg ctctgcccgc
     3541 cccggggtgg gcgccggggg gaccttaaag cctctgcccc ccaaggagcc cttcccagac
     3601 agccgccggc acccaccgct ccgtgggacg atccccgaag ctctagagct ttattgcggt
     3661 agtttatcac agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt
     3721 aagctgcaga agttggtcgt gaggcactgg gcaggtaagt atcaaggtta caagacaggt
     3781 ttaaggagac caatagaaac tgggcttgtc gagacagaga agactcttgc gtttctgata
     3841 ggcacctatt ggtcttactg acatccactt tgcctttctc tccacaggtg tccactccca
     3901 gttcaattac agctcttaag gctagagtac ttaatacgac tcactatagg ctagcatggt
     3961 gcccaagaag aagaggaaag tctccaacct gctgactgtg caccaaaacc tgcctgccct
     4021 ccctgtggat gccacctctg atgaagtcag gaagaacctg atggacatgt tcagggacag
     4081 gcaggccttc tctgaacaca cctggaagat gctcctgtct gtgtgcagat cctgggctgc
     4141 ctggtgcaag ctgaacaaca ggaaatggtt ccctgctgaa cctgaggatg tgagggacta
     4201 cctcctgtac ctgcaagcca gaggcctggc tgtgaagacc atccaacagc acctgggcca
     4261 gctcaacatg ctgcacagga gatctggcct gcctcgccct tctgactcca atgctgtgtc
     4321 cctggtgatg aggagaatca gaaaggagaa tgtggatgct ggggagagag ccaagcaggc
     4381 cctggccttt gaacgcactg actttgacca agtcagatcc ctgatggaga actctgacag
     4441 atgccaggac atcaggaacc tggccttcct gggcattgcc tacaacaccc tgctgcgcat
     4501 tgccgaaatt gccagaatca gagtgaagga catctcccgc accgatggtg ggagaatgct
     4561 gatccacatt ggcaggacca agaccctggt gtccacagct ggtgtggaga aggccctgtc
     4621 cctgggggtt accaagctgg tggagagatg gatctctgtg tctggtgtgg ctgatgaccc
     4681 caacaactac ctgttctgcc gggtcagaaa gaatggtgtg gctgcccctt ctgccacctc
     4741 ccaactgtcc acccgggccc tggaagggat ctttgaggcc acccaccgcc tgatctatgg
     4801 tgccaaggat gactctgggc agagatacct ggcctggtct ggccactctg ccagagtggg
     4861 tgctgccagg gacatggcca gggctggtgt gtccatccct gaaatcatgc aggctggtgg
     4921 ctggaccaat gtgaacattg tgatgaacta catcagaaac ctggactctg agactggggc
     4981 catggtgagg ctgctcgagg atggggactg aaagcttatc gataccgagc gctgctcgag
     5041 agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg
     5101 ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact
     5161 aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg
     5221 ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac
     5281 aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc
     5341 ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt
     5401 agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct
     5461 acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg
     5521 tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     5581 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     5641 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     5701 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     5761 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     5821 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     5881 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5941 cctttagggt tc
//
