LOCUS       sequence-450390-        6000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac cargo vector encoding A to B pegRNA marked with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6000)
  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 6000)
  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: pTBL3241 PB-AtoBpegRNA-mCherry
FEATURES             Location/Qualifiers
     source          1..6000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..52)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        815..833
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   884..946
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    1529..1753
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1742..1759
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1792..2499)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2067..2086)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2336..2354
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     intron          2519..3457
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2542..2562)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(3458..3669)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        complement(4109..4357)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4167..4186)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4337..4357)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4953..4987
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5016..5035)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5017..5035)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(5147..5735)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5227..5246)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5949..5968)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(5960..6000,1..775)
                     /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(5960..6000,1..775)
                     /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"
ORIGIN
        1 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
       61 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
      121 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
      181 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
      241 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
      301 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
      361 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgcctcgctc aggcgcaatc
      421 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
      481 tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt
      541 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
      601 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
      661 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
      721 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
      781 attggttaat tggttgtaac acgcggccgc tcggaattaa ccctcactaa agggaacaaa
      841 agctggatta attaagcggc cgcatacgcg tatactagat taaccctaga aagataatca
      901 tattgtgacg tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct
      961 gtatatcgag gtttatttat taatttgaat agatattaag ttttattata tttacactta
     1021 catactaata ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa
     1081 caaaaactca aaatttcttc tataaagtaa caaaactttt aactagtgtt catccatacc
     1141 atgcgtgggt accccataga gcccaccgct gtaaggttag tgtgtccagc atgcaacctt
     1201 tccttcctgg atgagcatcc ctggaatagg agagccagcc tgcctcctgc gctggcacag
     1261 agcccggttc cctagacaac tgcctctcca aatctgatgt ccagcgccac ctggtgtcca
     1321 catcaagcag acacaattaa tagtcaacct gttcagggtt cgtaacatct ctgtaactgc
     1381 ttggactgtt gtgatatgcc atatatgcag tggtgttcgc cggcgctggt tccggctgtc
     1441 ttgcttagtt gtgagtcgcc aggcagtcat cggtatgatc tgtacatgat tcgtcagtgt
     1501 gagcaccact gacgagcaga tttccagccc atagagccca ccgcatcccc agcatgcctg
     1561 ctattgtctt cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa
     1621 tgacacctac tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga
     1681 gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa
     1741 ctagaaggca cagtcgaggc gtcgctcgag ttaggattcg acatctgtag acttgtacag
     1801 ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt tcgtactgtt ccacgatggt
     1861 gtagtcctcg ttgtgggagg tgatgtccaa cttgatgttg acgttgtagg cgccgggcag
     1921 ctgcacgggc ttcttggcct tgtaggtggt cttgacctca gcgtcgtagt ggccgccgtc
     1981 cttcagcttc agcctctgct tgatctcgcc cttcagggcg ccgtcctcgg ggtacatccg
     2041 ctcggaggag gcctcccagc ccatggtctt cttctgcatt acggggccgt cggaggggaa
     2101 gttggtgccg cgcagcttca ccttgtagat gaactcgccg tcctgcaggg aggagtcctg
     2161 ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg cgctcccact tgaagccctc
     2221 ggggaaggac agcttcaagt agtcgggaat gtcggcgggg tgcttcacgt aggccttgga
     2281 gccgtacatg aactgagggg acaggatgtc ccaggcgaag ggcagggggc cacccttggt
     2341 caccttcagc ttggcggtct gggtgccctc gtaggggcgg ccctcgccct cgccctcgat
     2401 ctcgaactcg tggccgttca cggagccctc catgtgcacc ttgaagcgca tgaactcctt
     2461 gatgatggcc atgttatcct cctcgccctt gctcaccata gtggctagct cacgacacct
     2521 gaaatggaag aaaaaaactt tgaaccactg tctgaggctt gagaatgaac caagatccaa
     2581 actcaaaaag ggcaaattcc aaggagaatt acatcaagtg ccaagctggc ctaacttcag
     2641 tctccaccca ctcagtgtgg ggaaactcca tcgcataaaa cccctccccc caacctaaag
     2701 acgacgtact ccaaaagcgc gagaactaat cgaggtgcct ggacggcgcc cggtactccg
     2761 tggagtcaca tgaagcgacg gctgaggacg gaaaggccct tttcctttgt gtgggtgact
     2821 cacccgcccg ctctcccgag cgccgcgtcc tccattttga gctccctgca gcagggccgg
     2881 gaagcggcca tctttccgct cacgcaactg gtgccgaccg ggccagcctt gccgcccagg
     2941 gcggggcgat acacggcggc gcgaggccag gcaccagagc aggccggcca gcttgagact
     3001 acccccgtcc gattctcggt ggccgcgctc gcaggccccg cctcgccgaa catgtgcgct
     3061 gggacgcacg ggccccgtcg ccgcccgcgg ccccaaaaac cgaaatacca gtgtgcagat
     3121 cttggcccgc atttacaaga ctatcttgcc agaaaaaaag cgtcgcagca ggtcatcaaa
     3181 aattttaaat ggctagagac ttatcgaaag cagcgagaca ggcgcgaagg tgccaccaga
     3241 ttcgcacgcg gcggccccag cgcccaggcc aggcctcaac tcaagcacga ggcgaagggg
     3301 ctccttaagc gcaaggcctc gaactctccc acccacttcc aacccgaagc tcgggatcaa
     3361 gaatcacgta ctgcagccag gtggaagtaa ttcaaggcac gcaagggcca taacccgtaa
     3421 agaggccagg cccgcgggaa ccacacacgg cacttacctg tgttctggcg gcaaacccgt
     3481 tgcgaaaaag aacgttcacg gcgactactg cacttatata cggttctccc ccaccctcgg
     3541 gaaaaaggcg gagccagtac acgacatcac tttcccagtt taccccgcgc caccttctct
     3601 aggcaccggt tcaattgccg acccctcccc ccaacttctc ggggactgtg ggcgatgtgc
     3661 gctctgccca ctgacgggca ccgtttgaaa actattccgg aatgggaatc caggggtagg
     3721 agatgctttg gctctagatg caagacatga agcgtttgct tctggccacc agggggagct
     3781 gctgggacgc agacggactc caaggactgg agccagaagc ctggtgttgt aactgttctg
     3841 agatcgtcgc ccgccagcgc agacaagctc ggtcacgcgt tcgactgtgt ggagctttat
     3901 tcacttcagc actccagtct ttctagaaga tggaaaaatc accatcatac cactgggcca
     3961 tctatcattg atggcagagg aaaggaagcc ctgcttcctc cagcaccgac tcggtgccac
     4021 tttttcaagt tgataacgga ctagccttat ttaaacttgc tatgctgttt ccagcttagc
     4081 tcttaaactc aatgatggtg atgatggcgg tgtttcgtcc tttccacaag atatataaag
     4141 ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata
     4201 attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta
     4261 atatctttgt gtttacagtc aaattaattc taattatctc tctaacagcc ttgtatcgta
     4321 tatgcaaata tgaaggaatc atgggaaata ggccctccgt tttaaccaac aacattgcaa
     4381 catatagttt tagtgtgatt cctttcattc agtgtttttc aagagtttta aaacaacacc
     4441 agctttccaa ttgtactgaa tttgtttttt tccctttcat caccacaaaa atccagtttt
     4501 gcccagtata tggggaaacc attcttactg aaccagcaga gggcgctgat gcactagtga
     4561 tgtccgatat gcctaagact taaaacatat agaaacatcg tctgcaatta tcggccagtt
     4621 cagtctagat gcgaattcag ggcccatgga gatctgggcc catgctagca ttctagttga
     4681 tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac
     4741 aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt
     4801 ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc
     4861 ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct
     4921 atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt
     4981 tctagggtta atctagaatg gcgcgcccca attcgcccta tagtgagtcg tattaattgg
     5041 cggccgcacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5101 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5161 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5221 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5281 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5341 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5401 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5461 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5521 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5581 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5641 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5701 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5761 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg actgatcctt
     5821 caactcagca aaagttcgat ttattcaaca aagccacgtt gtgtctcaaa atctctgatg
     5881 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     5941 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ccaggccgcg
//
