LOCUS       sequence-450374-        6266 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac cargo vector encoding B to A pegRNA marked with sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6266)
  AUTHORS   Loveless BT, Carlson CK, Dentzel Helmy CA, Hu VJ, Ross SK, Demelo 
            MC, Murtaza A, Liang G, Ficht M, Singhai A, Pajoh-Casco MJ, Liu CC
  TITLE     Open-ended molecular recording of sequential cellular events into 
            DNA
  JOURNAL   bioRxiv 2021.11.05.467507
REFERENCE   2  (bases 1 to 6266)
  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: "bioRxiv 
            2021.11.05.467507"
COMMENT     Sequence Label: pTBL3401 BtoApegRNA-sfGFP
FEATURES             Location/Qualifiers
     source          1..6266
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        635..653
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   704..766
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     1687..1706
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    1705..1760
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     1806..1825
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(1919..2632)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     intron          2652..3590
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2675..2695)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(3591..3802)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        complement(4195..4443)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4253..4272)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4423..4443)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   5039..5073
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5102..5121)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5103..5121)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(5233..5821)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5313..5332)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6035..6054)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(6046..6266,1..595)
                     /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             join(6046..6266,1..595)
                     /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     complement(6119..6138)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
       61 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
      121 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
      181 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgcctcgctc aggcgcaatc
      241 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
      301 tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt
      361 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
      421 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
      481 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
      541 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
      601 attggttaat tggttgtaac acgcggccgc tcggaattaa ccctcactaa agggaacaaa
      661 agctggatta attaagcggc cgcatacgcg tatactagat taaccctaga aagataatca
      721 tattgtgacg tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct
      781 gtatatcgag gtttatttat taatttgaat agatattaag ttttattata tttacactta
      841 catactaata ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa
      901 caaaaactca aaatttcttc tataaagtaa caaaactttt aactagtgtt catccatacc
      961 atgcgtgggt accccataga gcccaccgct gtaaggttag tgtgtccagc atgcaacctt
     1021 tccttcctgg atgagcatcc ctggaatagg agagccagcc tgcctcctgc gctggcacag
     1081 agcccggttc cctagacaac tgcctctcca aatctgatgt ccagcgccac ctggtgtcca
     1141 catcaagcag acacaattaa tagtcaacct gttcagggtt cgtaacatct ctgtaactgc
     1201 ttggactgtt gtgatatgcc atatatgcag tggtgttcgc cggcgctggt tccggctgtc
     1261 ttgcttagtt gtgagtcgcc aggcagtcat cggtatgatc tgtacatgat tcgtcagtgt
     1321 gagcaccact gacgagcaga tttccagcat tacgccaagc ttgggctgca ggtcgaggga
     1381 tctccataag agaagaggga cagctatgac tgggagtagt caggagagga ggaaaaatct
     1441 ggctagtaaa acatgtaagg aaaattttag ggatgttaaa gaaaaaaata acacaaaaca
     1501 aaatataaaa aaaatctaac ctcaagtcaa ggcttttcta tggaataagg aatggacagc
     1561 agggggctgt ttcatatact gatgacctct ttatagccaa cctttgttca tggcagccag
     1621 catatgggca tatgttgcca aactctaaac caaatactca ttctgatgtt ttaaatgatt
     1681 tgccctccca tatgtccttc cgagtgagag acacaaaaaa ttccaacaca ctattgcaat
     1741 gaaaataaat ttcctttatt agccagaagt cagatgctca aggggcttca tgatgtcccc
     1801 ataatttttg gcagagggaa aaagatctca gtggtatttg tgagccaggg cattggccac
     1861 accagccacc accttctgat aggcagcctg cacctgagga gtgaatctcg agttaggatt
     1921 tgtacagttc atccatacca tgcgtgatgc ccgctgcggt tacgaactcc agcagaacca
     1981 tatgatcgcg tttctcgttc ggatctttag acagaacgct ttgcgtgctc agatagtgat
     2041 tgtctggcag cagaacagga ccatcaccga ttggagtgtt ttgctggtag tgatcagcca
     2101 gctgcacgct gccatcctcc acgttgtggc gaattttaaa attcgcttta atgccatttt
     2161 tttgtttatc ggcggtgatg taaacattgt ggctgttaaa attgtattcc agcttatggc
     2221 ccaggatatt gccgtcctct ttaaagtcaa tgcctttcag ctcaatgcgg tttaccaggg
     2281 tatcgccttc aaatttcact tccgcacgcg ttttgtacgt gccgtcatcc ttaaaggaaa
     2341 tcgtgcgttc ctgcacatag ccttccggca tggcggactt gaagaagtca tgctgcttca
     2401 tatggtccgg ataacgagca aagcactgaa caccataagt cagcgtcgtt accagagtcg
     2461 gccaaggaac cggcagttta ccagtagtac agatgaactt cagcgtcagt ttaccattag
     2521 ttgcgtcacc ttcaccctcg ccacgcacgg aaaacttatg accgttgaca tcaccatcca
     2581 gttccaccag aatagggacg acaccagtga acagctcttc gcctttacgc atagtggcta
     2641 gctcacgaca cctgaaatgg aagaaaaaaa ctttgaacca ctgtctgagg cttgagaatg
     2701 aaccaagatc caaactcaaa aagggcaaat tccaaggaga attacatcaa gtgccaagct
     2761 ggcctaactt cagtctccac ccactcagtg tggggaaact ccatcgcata aaacccctcc
     2821 ccccaaccta aagacgacgt actccaaaag cgcgagaact aatcgaggtg cctggacggc
     2881 gcccggtact ccgtggagtc acatgaagcg acggctgagg acggaaaggc ccttttcctt
     2941 tgtgtgggtg actcacccgc ccgctctccc gagcgccgcg tcctccattt tgagctccct
     3001 gcagcagggc cgggaagcgg ccatctttcc gctcacgcaa ctggtgccga ccgggccagc
     3061 cttgccgccc agggcggggc gatacacggc ggcgcgaggc caggcaccag agcaggccgg
     3121 ccagcttgag actacccccg tccgattctc ggtggccgcg ctcgcaggcc ccgcctcgcc
     3181 gaacatgtgc gctgggacgc acgggccccg tcgccgcccg cggccccaaa aaccgaaata
     3241 ccagtgtgca gatcttggcc cgcatttaca agactatctt gccagaaaaa aagcgtcgca
     3301 gcaggtcatc aaaaatttta aatggctaga gacttatcga aagcagcgag acaggcgcga
     3361 aggtgccacc agattcgcac gcggcggccc cagcgcccag gccaggcctc aactcaagca
     3421 cgaggcgaag gggctcctta agcgcaaggc ctcgaactct cccacccact tccaacccga
     3481 agctcgggat caagaatcac gtactgcagc caggtggaag taattcaagg cacgcaaggg
     3541 ccataacccg taaagaggcc aggcccgcgg gaaccacaca cggcacttac ctgtgttctg
     3601 gcggcaaacc cgttgcgaaa aagaacgttc acggcgacta ctgcacttat atacggttct
     3661 cccccaccct cgggaaaaag gcggagccag tacacgacat cactttccca gtttaccccg
     3721 cgccaccttc tctaggcacc ggttcaattg ccgacccctc cccccaactt ctcggggact
     3781 gtgggcgatg tgcgctctgc ccactgacgg gcaccgtttg aaaactattc cggaatggga
     3841 atccaggggt aggagatgct ttggctctag atgcaagaca tgaagcgttt gcttctggcc
     3901 accaggggga gctgctggga cgcagacgga ctccaaggac tggagccaga agcctggtgt
     3961 tgcgactgtg tggagcttta ttcacttcag cactccagtc tttctagaag atggaaaaag
     4021 ggccatctat catgtggcca tcatcaccat cattgatggc agaggaaagg aagccctgct
     4081 tcctccagca ccgactcggt gccacttttt caagttgata acggactagc cttatttaaa
     4141 cttgctatgc tgtttccagc ttagctctta aactcaatga tagatggccc agtcggtgtt
     4201 tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag
     4261 catatgatag tccattttaa aacataattt taaaactgca aactacccaa gaaattatta
     4321 ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattctaat
     4381 tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc
     4441 ctccgtttta accaacaaca ttgcaacata tagttttagt gtgattcctt tcattcagtg
     4501 tttttcaaga gttttaaaac aacaccagct ttccaattgt actgaatttg tttttttccc
     4561 tttcatcacc acaaaaatcc agttttgccc agtatatggg gaaaccattc ttactgaacc
     4621 agcagagggc gctgatgcac tagtgatgtc cgatatgcct aagacttaaa acatatagaa
     4681 acatcgtctg caattatcgg ccagttcagt ctagatgcga attcagggcc catggagatc
     4741 tgggcccatg ctagcattct agttgatatc tataacaaga aaatatatat ataataagtt
     4801 atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa tcttaaaagt
     4861 cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt caaaatcagt
     4921 gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac tgagatgtcc
     4981 taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt caagaatgca
     5041 tgcgtcaatt ttacgcagac tatctttcta gggttaatct agaatggcgc gccccaattc
     5101 gccctatagt gagtcgtatt aattggcggc cgcacggtta tccacagaat caggggataa
     5161 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5221 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5281 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5341 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5401 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5461 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5521 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5581 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5641 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5701 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5761 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5821 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5881 agggattttg gtcatgactg atccttcaac tcagcaaaag ttcgatttat tcaacaaagc
     5941 cacgttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     6001 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     6061 cgggaaacgt cttgctccag gccgcgatta aattccaaca tggatgctga tttatatggg
     6121 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg
     6181 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     6241 acagatgaga tggtcagact aaactg
//
