LOCUS       sequence-477255-        7766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Updated ENGRAM plasmid that is ready for both 5' and 3'-FT ENGRAM 
            cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7766)
  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 7766)
  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: P021-mPB-ENGRAM-All-in-one-PGK-BFP-P2A-Blast
FEATURES             Location/Qualifiers
     source          1..7766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..67
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    545..559
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     regulatory      562..571
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             568..1128
                     /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             1135..1155
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    1296..1310
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     primer_bind     complement(1358..1375)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1364..1588
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1642..2152
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2071..2089
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      2163..2172
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2889..2945
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2946..3344
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(3405..3425)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3467..3588
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3504..3523)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3558..3577
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4351..4385
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4876..4892)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4876..4892)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4889..4911)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4900..4916
                     /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(4924..4930,4931..4948,4949..4954))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4969..4990
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5107..5124)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5278..5866)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5358..5377)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6037..6828,6829..6897))
                     /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     6660..6679
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6898..7002)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7028..7483)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7165..7186)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7377..7396
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7610..7632
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7624..7641
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7625..7641
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg tgtaaaattg
       61 acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa
      121 gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt
      181 tatttattta ttaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt
      241 atgagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
      301 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
      361 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
      421 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaaccggt
      481 gagggcgtcg caggacagct ggatcccacg aactgagacc attaaatgac agcatgggtc
      541 tcaactgccg tataggcagc cgccaccatg gaccactacc tcgacattcg cttgcgaccg
      601 gacccggaat ttcccccggc gcaactcatg agcgtgctct tcggcaagct ccaccaggcc
      661 ctggtggcac agggcgggga caggatcggc gtgagcttcc ccgacctcga cgaaagccgc
      721 tcccggctgg gcgagcgcct gcgcattcat gcctcggcgg acgaccttcg tgccctgctc
      781 gcccggccct ggctggaagg gttgcgggac catctgcaat tcggagaacc ggcagtcgtg
      841 cctcacccca caccgtaccg tcaggtcagt cgggttcagg cgaaaagcaa tccggaacgc
      901 ctgcggcggc ggctcatgcg ccggcacgat ctgagtgagg aggaggctcg gaaacgcatt
      961 cccgatacgg tcgcgagagc cttggacctg cccttcgtca cgctacgcag ccagagcacc
     1021 ggacagcact tccgtctgtt catccgccac gggccgttgc aggtgacggc agaggaagga
     1081 ggattcacct gttacgggtt gagcaaagga ggtttcgttc cctggttcgg gtcccccaag
     1141 aagaagagga aagtctaatc agtagggttg taaaggtttt tcttttcctg agaaaacaac
     1201 cttttgtttt ctcaggtttt gctttttggc ctttccctag ctttaaaaaa aaaaaagcaa
     1261 aactcaccga ggcagttcgt atcggtctgc gaacactgcc gtataggcag cacgtcttca
     1321 ttaaatgaca gctggaagac atagtccgaa acccgatcct cgactgtgcc ttctagttgc
     1381 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     1441 actgtccttt cctaataaaa tgagaaaatt gcatcgcatt gtctgagtag gtgtcattct
     1501 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaca caatagcagg
     1561 catgctgggg atgcggtggg ctctatgggg atcgctttcc tctgaacgct tctcgctgct
     1621 ctttgagcca tggcagacac cggggttggg gttgcgcctt ttccaaggca gccctgggtt
     1681 tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg
     1741 gtcacgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt
     1801 gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc
     1861 ggcgtgccgg acgtgacaaa cggaagccgc acctctcact agtaccctcg cagacggaca
     1921 gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata gcggctgctc
     1981 agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg ggtgtggggc
     2041 ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg
     2101 cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc aggctagcca
     2161 ccgccaccat ggtttccaag ggcgaagaac tgatcaaaga aaacatgcac atgaagctct
     2221 acatggaagg caccgtggac aaccaccact tcaagtgtac cagcgaaggc gagggcaagc
     2281 cttacgaggg cacccagacc atgagaatca aggtggtgga aggcggccct ctgcctttcg
     2341 cctttgatat cctggccacc agctttctgt acggcagcaa gaccttcatc aatcacaccc
     2401 agggcatccc cgatttcttc aagcagagct tccccgaggg cttcacctgg gagagagtga
     2461 ccacatacga ggatggcggc gtgctgacag ccacacagga tacaagcctc caggacggct
     2521 gcctgatcta caacgtgaag atccggggcg tgaacttcac cagcaacggc cccgtgatgc
     2581 agaagaaaac ccttggctgg gaagccttca ccgagacact gtatcctgcc gatggcggcc
     2641 tggaaggcag aaacgatatg gccctgaagc tcgtcggcgg ctctcacctg attgccaatg
     2701 ccaagaccac ctacagaagc aagaagcccg ccaagaacct gaagatgccc ggcgtgtact
     2761 acgtggacta ccggctggaa agaatcaaag aggccaacaa cgagacatac gtggaacagc
     2821 acgaggtggc cgtggccaga tactgtgatc tgccttctaa gctgggccac aagctgaacg
     2881 gatctggagc tactaacttc agcctgctga agcaggctgg cgacgtggag gagaaccctg
     2941 gacccatggc caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
     3001 caatcaacag catccccatc tctgaagatt acagcgtcgc cagcgcagct ctctctagcg
     3061 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
     3121 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
     3181 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
     3241 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
     3301 ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaacgtcga caatcaacct
     3361 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     3421 ctatgtggat acgctgcttt aatgcctttg tatcatgcgt taactaaact tgtttattgc
     3481 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3541 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggaa
     3601 ttgactcaaa tgatgtcaat tagtctatca gaagctcatc tggactccct tccgggggac
     3661 aagacatccc tgtttaatat ttaaacagca gtgttcccaa actgggttct tatatccctt
     3721 gctctggtca accaggttgc agggtttcct gtcctcacag gaacgaagtc cctaaagaaa
     3781 cagtggcagc caggtttagc cccggaattg actggattcc ttttttaggg cccattggta
     3841 tggctttttc cccgtatccc cccaggtgtc tgcaggctca aagagcagcg agaagcgttc
     3901 agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca cgctgccggc
     3961 tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg ctgctgcccc
     4021 ctagcggggg agggacgtaa ttacatccct gggggctttg ggggggggct gtccctgata
     4081 tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa
     4141 tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt
     4201 gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct
     4261 cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat
     4321 ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc
     4381 tagggttaat ctagctgcat caggatcata tcgtcgggtc ttttttccgg ctcagtcatc
     4441 gcccaagctg gcgctatctg ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg
     4501 ttgaagcggc atggaaagag tttgccgagg atgactgctg ctgcattgac gttgagcgaa
     4561 aacgcacgtt taccatgatg attcgggaag gtgtggccat gcacgccttt aacggtgaac
     4621 tgttcgttca ggccacctgg gataccagtt cgtcgcggct tttccggaca cagttccgga
     4681 tggtcagccc gaagcgcatc agcaacccga acaataccgg cgacagccgg aactgccgtg
     4741 ccggtgtgca gattaatgac agcggtgcgg cgctgggata ttacgtcagc gaggacgggt
     4801 atcctggctg gatgccgcag aaatggacat ggataccccg tgagttaccc ggcgggcgcg
     4861 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4921 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4981 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5041 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5101 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5161 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5221 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5281 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5341 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5401 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5461 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5521 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5581 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5641 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5701 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5761 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5821 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5881 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5941 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6001 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6061 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6121 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6181 tccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6241 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6301 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6361 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6421 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6481 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6541 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6601 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6661 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6721 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6781 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6841 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6901 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6961 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7021 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     7081 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     7141 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     7201 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     7261 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     7321 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     7381 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     7441 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     7501 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     7561 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     7621 cgttgtaaaa cgacggccag tgagcgcgcc tcgttcattc acgtttttga acccgtggag
     7681 gacgggcaga ctcgcggtgc aaatgtgttt tacagcgtga tggagcagat gaagatgctc
     7741 gacacgctgc agaacacgca gctaga
//
