LOCUS       sequence-356957-        5681 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac plasmid for 5' ENGRAM recorder.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5681)
  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 5681)
  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-5'-ENGRAM
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(529..1320,1321..1389))
                     /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     1152..1171
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1390..1494)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1520..1975)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1657..1678)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1869..1888
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2102..2124
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2116..2133
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2117..2133
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2263..2325
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2766..2797
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      2863..2872
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2869..3429
                     /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             3436..3456
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    3597..3611
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     polyA_signal    3640..3761
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3677..3696)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3731..3750
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4524..4558
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5049..5065)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5049..5065)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5062..5084)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5073..5089
                     /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(5097..5103,5104..5121,5122..5127))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5142..5163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5280..5297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5451..5681,1..358))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5531..5550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
       61 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
      121 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
      181 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
      241 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
      301 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
      361 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
      421 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
      481 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
      541 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
      601 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
      661 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
      721 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      781 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      841 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      901 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      961 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     1021 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     1081 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     1141 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     1201 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     1261 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     1321 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     1381 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     1441 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     1501 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     1561 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     1621 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     1681 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     1741 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     1801 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     1861 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     1921 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     1981 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     2041 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     2101 tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cctcgttcat tcacgttttt
     2161 gaacccgtgg aggacgggca gactcgcggt gcaaatgtgt tttacagcgt gatggagcag
     2221 atgaagatgc tcgacacgct gcagaacacg cagctagatt aaccctagaa agataatcat
     2281 attgtgacgt acgttaaaga taatcatgtg taaaattgac gcatgtgttt tatcggtctg
     2341 tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat ttacacttac
     2401 atactaataa taaattcaac aaacaattta tttatgttta tttatttatt aaaaaaaaca
     2461 aaaactcaaa atttcttcta taaagtaaca aaacttttat gagggacagc ccccccccaa
     2521 agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg
     2581 gctccgctcc ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc
     2641 cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga
     2701 gcctgcagac acctgggggg atacggggaa aaggcctcca cggccagctc tagacctgca
     2761 ggcactagag ggtatataat ggaagctcga cttccagctt ggcaatccgg tactgtaccg
     2821 gtcacttgag acgattaaat ggacagcatg cgtctcaagt ccgccaccat ggaccactac
     2881 ctcgacattc gcttgcgacc ggacccggaa tttcccccgg cgcaactcat gagcgtgctc
     2941 ttcggcaagc tccaccaggc cctggtggca cagggcgggg acaggatcgg cgtgagcttc
     3001 cccgacctcg acgaaagccg ctcccggctg ggcgagcgcc tgcgcattca tgcctcggcg
     3061 gacgaccttc gtgccctgct cgcccggccc tggctggaag ggttgcggga ccatctgcaa
     3121 ttcggagaac cggcagtcgt gcctcacccc acaccgtacc gtcaggtcag tcgggttcag
     3181 gcgaaaagca atccggaacg cctgcggcgg cggctcatgc gccggcacga tctgagtgag
     3241 gaggaggctc ggaaacgcat tcccgatacg gtcgcgagag ccttggacct gcccttcgtc
     3301 acgctacgca gccagagcac cggacagcac ttccgtctgt tcatccgcca cgggccgttg
     3361 caggtgacgg cagaggaagg aggattcacc tgttacgggt tgagcaaagg aggtttcgtt
     3421 ccctggttcg gatcccccaa gaagaagagg aaagtctgat cagtagggtt gtaaaggttt
     3481 ttcttttcct gagaaaacaa ccttttgttt tctcaggttt tgctttttgg cctttcccta
     3541 gctttaaaaa aaaaaaagca aaactcaccg aggcagttcg tatcggtctg cgaacactgc
     3601 cgtataggca gcgtcgaggg acctaataac ttcgaactaa acttgtttat tgcagcttat
     3661 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3721 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gaattgactc
     3781 aaatgatgtc aattagtcta tcagaagctc atctggtctc ccttccgggg gacaagacat
     3841 ccctgtttaa tatttaaaca gcagtgttcc caaactgggt tcttatatcc cttgctctgg
     3901 tcaaccaggt tgcagggttt cctgtcctca caggaacgaa gtccctaaag aaacagtggc
     3961 agccaggttt agccccggaa ttgactggat tcctttttta gggcccattg gtatggcttt
     4021 ttccccgtat ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa
     4081 agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga
     4141 tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg
     4201 gggagggacg taattacatc cctgggggct ttgggggggg gctgtccctg atatctataa
     4261 caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt
     4321 atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac
     4381 gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga
     4441 gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa
     4501 gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt
     4561 aatctagctg catcaggatc atatcgtcgg gtcttttttc cggctcagtc atcgcccaag
     4621 ctggcgctat ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg aggttgaagc
     4681 ggcatggaaa gagtttgccg aggatgactg ctgctgcatt gacgttgagc gaaaacgcac
     4741 gtttaccatg atgattcggg aaggtgtggc catgcacgcc tttaacggtg aactgttcgt
     4801 tcaggccacc tgggatacca gttcgtcgcg gcttttccgg acacagttcc ggatggtcag
     4861 cccgaagcgc atcagcaacc cgaacaatac cggcgacagc cggaactgcc gtgccggtgt
     4921 gcagattaat gacagcggtg cggcgctggg atattacgtc agcgaggacg ggtatcctgg
     4981 ctggatgccg cagaaatgga catggatacc ccgtgagtta cccggcgggc gcgcttggcg
     5041 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5101 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5161 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5221 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5281 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5341 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5401 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5461 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5521 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5581 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5641 tctcatagct cacgctgtag gtatctcagt tcggtgtagg t
//
