LOCUS       sequence-172437-        7386 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid containing the BrokenHeart construct: a 
            hyperpiggybac donor transposon interrupting the tdTomato 
            fluorophore. Transposase activity rescues coding sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7386)
  AUTHORS   Cammack AJ, Moudgil A, Chen J, Vasek MJ, Shabsovich M, McCullough K,
            Yen A, Lagunas T, Maloney SE, He J, Chen X, Hooda M, Wilkinson MN, 
            Miller TM, Mitra RD, Dougherty JD
  TITLE     A viral toolkit for recording transcription factor-DNA interactions 
            in live mouse tissues.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 May 5;117(18):10003-10014. doi: 
            10.1073/pnas.1918241117. Epub 2020 Apr 16.
  PUBMED    32300008
REFERENCE   2  (bases 1 to 7386)
  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: 
            "10.1073/pnas.1918241117"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2020-05-5- 5"; volume: "117"; issue: "18"; pages: 
            "10003-10014"
COMMENT     Sequence Label: AAV-short_BrokenHeart (no BC)
FEATURES             Location/Qualifiers
     source          1..7386
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   374..503
                     /label=AAV2 ITR
     primer_bind     complement(539..558)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(540..558)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(569..586)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(569..585)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(578..600)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        985..1048
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1049..1117,1118..1909)
                     /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(1267..1286)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2080..2668
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2569..2588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2725..2768)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     repeat_region   2950..3079
                     /label=AAV2 ITR
     polyA_signal    3117..3324
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3313..3330
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     protein_bind    complement(3379..3412)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(3463..3496)
                     /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    3512..3536
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        3609..4791
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3840..4782
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4739..4759
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4805..4814
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     repeat_region   5194..5256
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5557..5573)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5570..5592)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5581..5597
                     /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(5605..5611,5612..5629,5630..5635))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5650..5671
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   6130..6164
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     6184..6203
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6378..6401
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    6584..6705
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6621..6640)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(6962..6981)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     protein_bind    complement(7101..7125)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    7138..7171
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    7222..7255
                     /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)."
ORIGIN
        1 cccccccccc gcccccatcg ctgcacaaaa taattaaaaa ataaataaat acaaaattgg
       61 gggtggggag gggggggaga tggggagagt gaagcagaac gtggggctca cctcgaccat
      121 ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat
      181 gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact
      241 tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca
      301 ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca ttatttagcg
      361 atctgacgtc aataggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
      421 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
      481 tcagtgagcg agcgagcgcg cagccatgcg gccttgacgg ccttccgcct acaattcgcc
      541 ctatagtgag tcgtattacg tcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
      601 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
      661 gtaatagcga agaggcccgc accgaaacgc ccttcccaac agttgcgcag cctgaatggc
      721 gaatgggagc gccctgtagc ggccactcaa ccctatctcg gtctattctt ttgatttata
      781 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
      841 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
      901 cgcggaaccc ctatttgttt atttttctaa atacaccaaa taaaaaggcc tgcgattacc
      961 agcaggcctg ttattagctt caaatatgta tccgctcatg agacaataac cctgataaat
     1021 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     1081 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     1141 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1201 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1261 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1321 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1381 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1441 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1501 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1561 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1621 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1681 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1741 taaatctgga gccggtgagc gtggttctcg cggtatcatt gcagcactgg ggccagatgg
     1801 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1861 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1921 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1981 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     2041 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2101 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2161 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2221 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2281 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2341 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2401 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2461 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2521 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2581 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2641 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2701 ggccttttgc tggccttttg ctcaataaaa cgaaaggctc agtcggaaga ctgggccttt
     2761 cgttttattt aggcacccca ggctttaccc gaacgaccga gcgcagcgag tcagtgagcg
     2821 aggaagcgga gagcgcccaa tacgcaagga aacagctatg accatgttaa tgcagctggc
     2881 acgacaggtt tcccgactgg aaagcgggca gtgataggcg gaaggcccat gaggcccagt
     2941 ctctacatgc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg
     3001 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
     3061 tccatcacta ggggttcctt cgatcacacg atcgactctc tatagagccc accgcatccc
     3121 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
     3181 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
     3241 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
     3301 atggctggca actagaaggc acagtcgagg ctgatcagcg gtttagtaga ccagtgattt
     3361 aaatgctctg gagataaaat aacttcgtat agcatacatt atacgaagtt attctttgca
     3421 ccattctaaa gaataacagt gataatttct gggttaaggc aaataacttc gtataggata
     3481 ctttatacga agttatctgc acttatgcta gacaagtttg tacaaaaaag caggctcttt
     3541 aaaggaacca attcagtcga ctggatcccc gctctcgtca tcactgaggt ggagaagagc
     3601 atgcgtgagg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
     3661 agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact
     3721 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
     3781 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
     3841 taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc
     3901 cttgaattac ttccacgccc ctggctgcag tacgtgattc ttgatcccga gcttcgggtt
     3961 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga
     4021 gttgaggcct ggcttgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc
     4081 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg
     4141 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg
     4201 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg
     4261 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct
     4321 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc
     4381 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc
     4441 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa
     4501 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc
     4561 aggcacctcg attagttctt cagcttttgg agtacgtcgt ctttaggttg gggggagggg
     4621 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg
     4681 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc
     4741 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg aggatccact
     4801 agtcgccacc atggatagca ctgagaacgt catcaagccc ttcatgcgct tcaaggtgca
     4861 catggagggc tccgtgaacg gccacgagtt tgagatcgag ggcgagggcg agggcaagcc
     4921 ctacgagggc acccagaccg ccaagctgca ggtgaccaag ggcggccccc tgcccttcgc
     4981 ctgggacatc ctgtcccccc agttcatgta tggctccaag gtgtatacca agcaccccgc
     5041 cgacatcccc gactacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     5101 gaactttgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcac
     5161 cttcatctac cacgtgaagt tcatcggcgt taaccctaga aagataatca tattgtgacg
     5221 tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
     5281 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     5341 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca
     5401 aaatttcttc tataaagtaa caaaactttt ctgagcagcc cgggcacgtc atccggagat
     5461 catcctgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     5521 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5581 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5641 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5701 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5761 tttgcgtatt gggcgctctt ccgctggggg atctcgagta ccggagatcg gaagagcaca
     5821 cgtctgaact ccagtcacgg gcccgatcgc gataagctat ctataacaag aaaatatata
     5881 tataataagt tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa
     5941 atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt
     6001 tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga
     6061 ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt
     6121 tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaact tcccctccga
     6181 cggccccgta atgcagaaga agactctggg ctgggagccc tccaccgagc gcctgtatcc
     6241 ccgcgacggc gtgctgaagg gcgagatcca caaggcgctg aagctgaagg gcggcggcca
     6301 ctacctgtgt gagttcaagt caatctacat ggccaagaag cccgtgaagc tgccaggcta
     6361 ctactacgtg gactccaagc tggacatcac ctcccacaac gaggactaca ccgtggtgga
     6421 gcagtatgag cgcaccgagg cccgccacca cctgttccag tagcggccgc gactctagat
     6481 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     6541 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     6601 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     6661 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa
     6721 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     6781 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     6841 gtgccgccgc cttctatgaa aggttgggct tcggaatcgt tttcagggac gcctgctgga
     6901 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     6961 ctgaaacacg gaaggagaca atacaggaag gaacccgcgc tatgacggca ataaaaagac
     7021 agaataaaac gcacggtgtt gcctcaggtt actcatatat actaatcagg ggataacgca
     7081 ggaaagctcg agatatctag acccagcttt cttgtacaaa gtggtacttt agttgccata
     7141 acttcgtata atgtatgcta tacgaagtta ttaagaggtt tcatattgct aatagcagct
     7201 acaatccagc taccattctg cataacttcg tataaagtat cctatacgaa gttatagagg
     7261 tttgcagcga ctccccgccc gccgcgcgct tcgcttttta tagggccgcc gccgccgccg
     7321 cctcgccata aaaggaaact ttcggagcgc gccgctctga ttggctgccg ccgcacctct
     7381 ccgcct
//
