LOCUS       sequence-400595-        7151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid containing H2B fused piggyBac self-reporting 
            transposon with tdTomato marker for mammalian calling cards.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7151)
  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 7151)
  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-H2B-tdTomato-SRT
FEATURES             Location/Qualifiers
     source          1..7151
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     103..121
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(159..181)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     281..300
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     494..516
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     508..525
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     509..525
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   821..883
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1125..2307
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1356..2298
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2255..2275
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2321..2698
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      2711..2720
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2717..4144
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQAA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     CDS             4145..4165
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4169..4189
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4193..4213
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     repeat_region   4598..4632
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     repeat_region   4940..5069
                     /label=AAV2 ITR
     primer_bind     complement(5105..5121)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5118..5140)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5129..5145
                     /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(5153..5159,5160..5177,5178..5183))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5198..5219
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5507..6095)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5587..5606)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6266..7081)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6266..7081)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6989..7008
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(join(7082..7151,1..35))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
       61 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
      121 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
      181 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      241 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      301 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
      361 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
      421 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
      481 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt
      541 gggattgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac
      601 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
      661 cactaggggt tcctattgac gtcagatcgc taaataatga cgtatgttcc catagtaacg
      721 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      781 gcagtacatc aagtgtatca tacgtcaagg ccgcatttaa ccctagaaag ataatcatat
      841 tgtgacgtac gttaaagata atcatgtgta aaattgacgc atgtgtttta tcggtctgta
      901 tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt acacttacat
      961 actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaacaaa
     1021 aactcaaaat ttcttctata aagtaacaaa acttttatcg aattcctgca gcccagcttg
     1081 atcttaggtc cctcgacgaa ttctctagag aagagcatgc gtgaggctcc ggtgcccgtc
     1141 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     1201 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     1261 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     1321 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
     1381 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acgcccctgg
     1441 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg
     1501 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggct tgggcgctgg
     1561 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
     1621 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt
     1681 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac
     1741 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
     1801 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg
     1861 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg
     1921 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
     1981 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     2041 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc
     2101 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     2161 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     2221 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     2281 ttttttcttc catttcaggt gtcgtgaaaa ctacgccacc atgccagagc cagcgaagtc
     2341 tgctcccgcc ccgaaaaagg gctccaagaa ggcggtgact aaggcgcaga agaaaggcgg
     2401 caagaagcgc aagcgcagcc gcaaggagag ctattccatc tatgtgtaca aggttctgaa
     2461 gcaggtccac cctgacaccg gcatttcgtc caaggccatg ggcatcatga attcgtttgt
     2521 gaacgacatt ttcgagcgca tcgcaggtga ggcttcccgc ctggcgcatt acaacaagcg
     2581 ctcgaccatc acctccaggg agatccagac ggccgtgcgc ctgctgctgc ctggggagtt
     2641 ggccaagcac gccgtgtccg agggtactaa ggccatcacc aagtacacca gcgctaagga
     2701 tccaccggtc gccaccatgg tgagcaaggg cgaggaggtc atcaaagagt tcatgcgctt
     2761 caaggtgcgc atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga
     2821 gggccgcccc tacgagggca cccaggccgc caagctgaag gtgaccaagg gcggccccct
     2881 gcccttcgcc tgggacatcc tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa
     2941 gcaccccgcc gacatccccg attacaagaa gctgtccttc cccgagggct tcaagtggga
     3001 gcgcgtgatg aacttcgagg acggcggtct ggtgaccgtg acccaggact cctccctgca
     3061 ggacggcacg ctgatctaca aggtgaagat gcgcggcacc aacttccccc ccgacggccc
     3121 cgtaatgcag aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga
     3181 cggcgtgctg aagggcgaga tccaccaggc cctgaagctg aaggacggcg gccactacct
     3241 ggtggagttc aagaccatct acatggccaa gaagcccgtg caactgcccg gctactacta
     3301 cgtggacacc aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     3361 cgagcgctcc gagggccgcc accacctgtt cctggggcat ggcaccggca gcaccggcag
     3421 cggcagctcc ggcaccgcct cctccgagga caacaacatg gccgtcatca aagagttcat
     3481 gcgcttcaag gtgcgcatgg agggctccat gaacggccac gagttcgaga tcgagggcga
     3541 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg
     3601 ccccctgccc ttcgcctggg acatcctgtc cccccagttc atgtacggct ccaaggcgta
     3661 cgtgaagcac cccgccgaca tccccgatta caagaagctg tccttccccg agggcttcaa
     3721 gtgggagcgc gtgatgaact tcgaggacgg cggtctggtg accgtgaccc aggactcctc
     3781 cctgcaggac ggcacgctga tctacaaggt gaagatgcgc ggcaccaact tcccccccga
     3841 cggccccgta atgcagaaga agaccatggg ctgggaggcc tccaccgagc gcctgtaccc
     3901 ccgcgacggc gtgctgaagg gcgagatcca ccaggccctg aagctgaagg acggcggcca
     3961 ctacctggtg gagttcaaga ccatctacat ggccaagaag cccgtgcaac tgcccggcta
     4021 ctactacgtg gacaccaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga
     4081 acagtacgag cgctccgagg gccgccacca cctgttcctg tacggcatgg acgagctgta
     4141 caagccaaaa aagaagagaa aggtagatcc aaaaaagaag agaaaggtag atccaaaaaa
     4201 gaagagaaag gtataatcga taacaactcg agtaccggct taagtgatag atcggaagag
     4261 cggttcagca ggaatgccga gaccggttta aactcagtgt ccatagcgac gccggcgcga
     4321 taagctatct ataacaagaa aatatatata taataagtta tcacgtaagt agaacatgaa
     4381 ataacaatat aattatcgta tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg
     4441 tcattttgac tcacgcggtc gttatagttc aaaatcagtg acacttaccg cattgacaag
     4501 cacgcctcac gggagctcca agcggcgact gagatgtcct aaatgcacag cgacggattc
     4561 gcgctattta gaaagagaga gcaatatttc aagaatgcat gcgtcaattt tacgcagact
     4621 atctttctag ggttaagagc tcctgagatg caggtacatc ccactgatga gtcccaaata
     4681 ggacgaaacg cgcttcggtg cgtctgggat tccactgcta tccacgcggc cgcacggcat
     4741 tacgttagac gtctaggcca caccaccctg aaggcgcctg ctcgcctctg atctgttgaa
     4801 gctaagcagg gtcggtcctg gttagtactt ggatgggact ccgcctggta atagccggta
     4861 ccgtaggctt gcatggtcat agctgtttcc ttgcgctggg gatgcggtgg gctctataga
     4921 gagtcgatcg tgtgatcgaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     4981 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     5041 gcggcctcag tgagcgagcg agcgcgcagc atcccaatgg cgcgccgagc ttggctcgag
     5101 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5161 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5221 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5281 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5341 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5401 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5461 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5521 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5581 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5641 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5701 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5761 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5821 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5881 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5941 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6001 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6061 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6121 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6181 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6241 tatatgagta aacttggtct gacagttaga aaaactcatc gagcatcaaa tgaaactgca
     6301 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     6361 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     6421 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     6481 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     6541 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     6601 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     6661 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     6721 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccaggga
     6781 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     6841 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     6901 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     6961 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     7021 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     7081 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7141 acatatttga a
//
