LOCUS       sequence-400604-        7267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid containing a constitutive (non Cre-dependent) 
            BrokenHeart construct: a piggyBac donor transposon interrupting 
            tdTomato. Transposase activity rescues coding sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7267)
  AUTHORS   Yen A, Mateusiak C, Sarafinovska S, Gachechiladze MA, Guo J, Chen X,
            Moudgil A, Cammack AJ, Hoisington-Lopez J, Crosby M, Brent MR, Mitra
            RD, Dougherty JD
  TITLE     Calling Cards: A Customizable Platform to Longitudinally Record 
            Protein-DNA Interactions Over Time in Cells and Tissues.
  JOURNAL   Curr Protoc. 2023 Sep;3(9):e883. doi: 10.1002/cpz1.883.
  PUBMED    37755132
REFERENCE   2  (bases 1 to 7267)
  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: "Curr 
            Protoc."; date: "2023-09"; volume: "3(9):e883. doi"; pages: " 
            10.1002/cpz1.883"
COMMENT     Sequence Label: pAAV-rBrokenheart
FEATURES             Location/Qualifiers
     source          1..7267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   18..147
                     /label=AAV2 ITR
     promoter        405..680
                     /label=chicken beta-actin promoter
     protein_bind    complement(701..734)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    750..774
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        847..2029
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1078..2020
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1977..1997
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2043..2052
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     repeat_region   2432..2494
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2795..2811)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2808..2830)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2819..2835
                     /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(join(2843..2849,2850..2867,2868..2873))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2888..2909
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   3368..3402
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     3422..3441
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3616..3639
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3822..3943
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3859..3878)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4200..4219)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     protein_bind    complement(4339..4363)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    4376..4409
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4458..4475)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4464..4671
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4709..4838
                     /label=AAV2 ITR
     terminator      5020..5063
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(5120..5708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5200..5219)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5879..6670,6671..6739))
                     /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     6502..6521
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6740..6803)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7188..7210
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7202..7219
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7203..7219
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7230..7249
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7230..7248
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ccgtcaaggc cgcatggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg
       61 ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag
      121 tggccaactc catcactagg ggttcctatt gacgtcagat cgctaaataa tgacgtatgt
      181 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      241 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      301 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      361 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
      421 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
      481 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggsgcgcg ccrggsgggg
      541 sggggsgggg sgrggggsgg ggsggggcga ggcggagagg tgcggcggca gccaatcaga
      601 gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa
      661 agcgaagcgc gcggcgggcg gggagtcgct gcaaacctct ataacttcgt ataggatact
      721 ttatacgaag ttatctgcac ttatgctaga caagtttgta caaaaaagca ggctctttaa
      781 aggaaccaat tcagtcgact ggatccccgc tctcgtcatc actgaggtgg agaagagcat
      841 gcgtgaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
      901 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
      961 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     1021 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
     1081 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct
     1141 tgaattactt ccacgcccct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg
     1201 aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt
     1261 tgaggcctgg cttgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg
     1321 tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct
     1381 ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt
     1441 ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg
     1501 gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc
     1561 tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg
     1621 gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc
     1681 aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag
     1741 ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag
     1801 gcacctcgat tagttcttca gcttttggag tacgtcgtct ttaggttggg gggaggggtt
     1861 ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca
     1921 cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa
     1981 gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gatccactag
     2041 tcgccaccat ggatagcact gagaacgtca tcaagccctt catgcgcttc aaggtgcaca
     2101 tggagggctc cgtgaacggc cacgagtttg agatcgaggg cgagggcgag ggcaagccct
     2161 acgagggcac ccagaccgcc aagctgcagg tgaccaaggg cggccccctg cccttcgcct
     2221 gggacatcct gtccccccag ttcatgtatg gctccaaggt gtataccaag caccccgccg
     2281 acatccccga ctacaagaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     2341 actttgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcacct
     2401 tcatctacca cgtgaagttc atcggcgtta accctagaaa gataatcata ttgtgacgta
     2461 cgttaaagat aatcatgcgt aaaattgacg catgtgtttt atcggtctgt atatcgaggt
     2521 ttatttatta atttgaatag atattaagtt ttattatatt tacacttaca tactaataat
     2581 aaattcaaca aacaatttat ttatgtttat ttatttatta aaaaaaaaca aaaactcaaa
     2641 atttcttcta taaagtaaca aaacttttct gagcagcccg ggcacgtcat ccggagatca
     2701 tcctgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     2761 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     2821 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     2881 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     2941 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3001 tgcgtattgg gcgctcttcc gctgggggat ctcgagtacc ggagatcgga agagcacacg
     3061 tctgaactcc agtcacgggc ccgatcgcga taagctatct ataacaagaa aatatatata
     3121 taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat
     3181 cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc
     3241 aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact
     3301 gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc
     3361 aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaacttc ccctccgacg
     3421 gccccgtaat gcagaagaag actctgggct gggagccctc caccgagcgc ctgtatcccc
     3481 gcgacggcgt gctgaagggc gagatccaca aggcgctgaa gctgaagggc ggcggccact
     3541 acctgtgtga gttcaagtca atctacatgg ccaagaagcc cgtgaagctg ccaggctact
     3601 actacgtgga ctccaagctg gacatcacct cccacaacga ggactacacc gtggtggagc
     3661 agtatgagcg caccgaggcc cgccaccacc tgttccagta gcggccgcga ctctagatca
     3721 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     3781 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     3841 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3901 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc
     3961 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     4021 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     4081 gccgccgcct tctatgaaag gttgggcttc ggaatcgttt tcagggacgc ctgctggatg
     4141 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     4201 gaaacacgga aggagacaat acaggaagga acccgcgcta tgacggcaat aaaaagacag
     4261 aataaaacgc acggtgttgc ctcaggttac tcatatatac taatcagggg ataacgcagg
     4321 aaagctcgag atatctagac ccagctttct tgtacaaagt ggtactttag ttgccataac
     4381 ttcgtataat gtatgctata cgaagttatt ttatctccag agcatttaaa tcactggtct
     4441 actaaaccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4501 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4561 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4621 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     4681 ctctatagag agtcgatcgt gtgatcgaag gaacccctag tgatggagtt ggccactccc
     4741 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4801 tttgcccggg cggcctcagt gagcgagcga gcgcgcagca tgtagagact gggcctcatg
     4861 ggccttccgc ctatcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4921 acatggtcat agctgtttcc ttgcgtattg ggcgctctcc gcttcctcgc tcactgactc
     4981 gctgcgctcg gtcgttcggg taaagcctgg ggtgcctaaa taaaacgaaa ggcccagtct
     5041 tccgactgag cctttcgttt tattgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5101 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5161 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5221 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5281 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5341 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5401 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5461 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5521 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5581 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5641 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5701 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5761 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5821 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5881 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5941 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6001 gtgctgcaat gataccgcga gaaccacgct caccggctcc agatttatca gcaataaacc
     6061 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6121 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6181 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6241 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6301 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6361 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6421 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6481 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6541 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6601 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6661 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6721 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6781 attgtctcat gagcggatac atatttgaag ctaataacag gcctgctggt aatcgcaggc
     6841 ctttttattt ggtgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6901 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     6961 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     7021 aatagaccga gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg
     7081 cgcaactgtt gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     7141 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     7201 ttgtaaaacg acggccagtg agcgcgacgt aatacgactc actatagggc gaattgtagg
     7261 cggaagg
//
