LOCUS       sequence-456767-        5679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac plasmid for 3'-FT ENGRAM recorder.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5679)
  AUTHORS   Chen W, Choi J, Li X, Nathans JF, Martin B, Yang W, Hamazaki N, Qiu 
            C, Lalanne JB, Regalado S, Kim H, Agarwal V, Nichols E, Leith A, Lee
            C, Shendure J
  TITLE     Symbolic recording of signalling and cis-regulatory element activity
            to DNA.
  JOURNAL   Nature. 2024 Aug;632(8027):1073-1081. doi: 
            10.1038/s41586-024-07706-4. Epub 2024 Jul 17.
  PUBMED    39020177
REFERENCE   2  (bases 1 to 5679)
  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: 
            "10.1038/s41586-024-07706-4"; journalName: "Nature"; date: 
            "2024-08"; volume: "632"; issue: "8027"; pages: "1073-1081"
COMMENT     Sequence Label: PB-3-FT-ENGRAM
FEATURES             Location/Qualifiers
     source          1..5679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     129..151
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     143..160
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     144..160
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   290..352
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        793..824
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     protein_bind    851..865
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     regulatory      867..876
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             873..1433
                     /codon_start=1
                     /product="CRISPR-associated endoribonuclease responsible 
                     for pre-crRNA processing in Pseudomonas aeruginosa 
                     (Haurwitz et al., 2010)"
                     /label=Csy4
                     /translation="MDHYLDIRLRPDPEFPPAQLMSVLFGKLHQALVAQGGDRIGVSFP
                     DLDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQ
                     AKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGP
                     LQVTAEEGGFTCYGLSKGGFVPWF"
     CDS             1440..1460
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1665..1786
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1702..1721)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1756..1775
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   2549..2583
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3074..3090)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3074..3090)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3087..3109)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3098..3114
                     /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(3122..3128,3129..3146,3147..3152))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3167..3188
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3305..3322)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3476..4064)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3556..3575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4235..5026,5027..5095))
                     /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     4858..4877
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5096..5200)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5226..5679,1..2))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5363..5384)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5575..5594
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
       61 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
      121 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcct cgttcattca
      181 cgtttttgaa cccgtggagg acgggcagac tcgcggtgca aatgtgtttt acagcgtgat
      241 ggagcagatg aagatgctcg acacgctgca gaacacgcag ctagattaac cctagaaaga
      301 taatcatatt gtgacgtacg ttaaagataa tcatgtgtaa aattgacgca tgtgttttat
      361 cggtctgtat atcgaggttt atttattaat ttgaatagat attaagtttt attatattta
      421 cacttacata ctaataataa attcaacaaa caatttattt atgtttattt atttattaaa
      481 aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa cttttatgag ggacagcccc
      541 cccccaaagc ccccagggat gtaattacgt ccctcccccg ctagggggca gcagcgagcc
      601 gcccggggct ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg
      661 gacagcccgg gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc
      721 tctttgagcc tgcagacacc tggggggata cggggaaaag gcctccacgg ccagctctag
      781 acctgcaggc actagagggt atataatgga agctcgactt ccagcttggc aatccggtac
      841 tgtaccggta ctgccgtata ggcagcgcca ccatggacca ctacctcgac attcgcttgc
      901 gaccggaccc ggaatttccc ccggcgcaac tcatgagcgt gctcttcggc aagctccacc
      961 aggccctggt ggcacagggc ggggacagga tcggcgtgag cttccccgac ctcgacgaaa
     1021 gccgctcccg gctgggcgag cgcctgcgca ttcatgcctc ggcggacgac cttcgtgccc
     1081 tgctcgcccg gccctggctg gaagggttgc gggaccatct gcaattcgga gaaccggcag
     1141 tcgtgcctca ccccacaccg taccgtcagg tcagtcgggt tcaggcgaaa agcaatccgg
     1201 aacgcctgcg gcggcggctc atgcgccggc acgatctgag tgaggaggag gctcggaaac
     1261 gcattcccga tacggtcgcg agagccttgg acctgccctt cgtcacgcta cgcagccaga
     1321 gcaccggaca gcacttccgt ctgttcatcc gccacgggcc gttgcaggtg acggcagagg
     1381 aaggaggatt cacctgttac gggttgagca aaggaggttt cgttccctgg ttcggatccc
     1441 ccaagaagaa gaggaaagtc tgatcagtag ggttgtaaag gtttttcttt tcctgagaaa
     1501 acaacctttt gttttctcag gttttgcttt ttggcctttc cctagcttta aaaaaaaaaa
     1561 agcaaaactc accgaggcag ttcgtatcgg tctgcgacac ttgagacgat taaatggaca
     1621 gcatgcgtct caagtccgtc gagggaccta ataacttcga actaaacttg tttattgcag
     1681 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1741 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggaatt
     1801 gactcaaatg atgtcaatta gtctatcaga agctcatctg gtctcccttc cgggggacaa
     1861 gacatccctg tttaatattt aaacagcagt gttcccaaac tgggttctta tatcccttgc
     1921 tctggtcaac caggttgcag ggtttcctgt cctcacagga acgaagtccc taaagaaaca
     1981 gtggcagcca ggtttagccc cggaattgac tggattcctt ttttagggcc cattggtatg
     2041 gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag
     2101 aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc
     2161 ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct
     2221 agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt ccctgatatc
     2281 tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata
     2341 taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga
     2401 ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca
     2461 cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt
     2521 agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta
     2581 gggttaatct agctgcatca ggatcatatc gtcgggtctt ttttccggct cagtcatcgc
     2641 ccaagctggc gctatctggg catcggggag gaagaagccc gtgccttttc ccgcgaggtt
     2701 gaagcggcat ggaaagagtt tgccgaggat gactgctgct gcattgacgt tgagcgaaaa
     2761 cgcacgttta ccatgatgat tcgggaaggt gtggccatgc acgcctttaa cggtgaactg
     2821 ttcgttcagg ccacctggga taccagttcg tcgcggcttt tccggacaca gttccggatg
     2881 gtcagcccga agcgcatcag caacccgaac aataccggcg acagccggaa ctgccgtgcc
     2941 ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat
     3001 cctggctgga tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgct
     3061 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3121 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     3181 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3241 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3301 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3361 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3421 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3481 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3541 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3601 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3661 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3721 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3781 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3841 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3901 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3961 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4021 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4081 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4141 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4201 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4261 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4321 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4381 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4441 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4501 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4561 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4621 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4681 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4741 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4801 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4861 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4921 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4981 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5041 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5101 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5161 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5221 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5281 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5341 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5401 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     5461 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     5521 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     5581 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     5641 caccacaccc gccgcgctta atgcgccgct acagggcgc
//
