LOCUS       sequence-485115-        7188 bp    DNA     circular SYN 02-SEP-2025
DEFINITION  Expresses p53 transcription fluorescence reporter in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7188)
  AUTHORS   Tsuruoka T, Goto Y, Aoki K
  TITLE     Opto-p53: A Light-Controllable Activation of p53 Signaling Pathway.
  JOURNAL   Cell Struct Funct. 2025 Jun 12. doi: 10.1247/csf.25017.
  PUBMED    40500178
REFERENCE   2  (bases 1 to 7188)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Struct
            Funct. 2025 Jun 12. doi: 10.1247/csf.25017."
COMMENT     Sequence Label: 
            pPB-p53RE(CDKN1A)-CMVmin-mScarlet-I-NLSx3-AU1-PGKpuro
FEATURES             Location/Qualifiers
     source          1..7188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    1092..1213
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1129..1148)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1183..1202
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(1229..1828)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1312..1332)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1809..1828
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(1901..1920)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3360..3397
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3394..3418
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             3496..4191
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(3629..3647)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3897..3916
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4192..4212
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4216..4236
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4240..4260
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4297..4521
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4549..4565)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4573..4589
                     /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(4597..4603,4604..4621,4622..4627))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4642..4663
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   4897..4959
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5000..5020)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5000..5018)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5039..5055)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5063..5079
                     /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(5087..5093,5094..5111,5112..5117))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5132..5153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5270..5287)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5441..6029)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5521..5540)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6200..6991,6992..7060))
                     /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     6823..6842
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7061..7165)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcatg ggcccatctc gacattgatt
     1021 attgactagt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     1081 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     1141 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     1201 tcaatgtatc ttatcatgtc tgggatccct aggcaccggg cttgcgggtc atgcaccagg
     1261 tgcgcggtcc ttcgggcacc tcgacgtcgg cggtgacggt gaagccgagc cgctcgtaga
     1321 aggggaggtt gcggggcgcg gaggtctcca ggaaggcggg caccccggcg cgctcggccg
     1381 cctccactcc ggggagcacg acggcgctgc ccagaccctt gccctggtgg tcgggcgaga
     1441 cgccgacggt ggccaggaac cacgcgggct ccttgggccg gtgcggcgcc aggaggcctt
     1501 ccatctgttg ctgcgcggcc agccgggaac cgctcaactc ggccatgcgc gggccgatct
     1561 cggcgaacac cgcccccgct tcgacgctct ccggcgtggt ccagaccgcc accgcggcgc
     1621 cgtcgtccgc gacccacacc ttgccgatgt cgagcccgac gcgcgtgagg aagagttctt
     1681 gcagctcggt gacccgctcg atgtggcggt ccggatcgac ggtgtggcgc gtggcggggt
     1741 agtcggcgaa cgcggcggcg agggtgcgta cggccctggg gacgtcgtcg cgggtggcga
     1801 ggcgcaccgt gggcttgtac tcggtcatgg atccggtaag ctagcttggg gtcgaaaggc
     1861 ccggagatga ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc gtgcagaatg
     1921 ccgggcctcc ggaggacctt cgggcgcccg ccccgcccct gagcccgccc ctgagcccgc
     1981 ccccggaccc accccttccc agcctctgag cccagaaagc gaaggagcaa agctgctatt
     2041 ggccgctgcc ccaaaggcct acccgcttcc attgctcagc ggtgctgtcc atctgcacga
     2101 gtctagtgag acgtgctact tccatttgtc acgtcctgca cgacgcgagc tgcggggcgg
     2161 gggggaactt cctgactagg ggaggagtag aaggtggcgc gaaggggcca ccaaagaacg
     2221 gagccggttg gcgcctaccg gtggatgtgg cccccccaaa gcccccaggg atgtaattac
     2281 gtccctcccc cgctaggggg cagcagcgag ccgcccgggg cggcgcggaa ttccctgctt
     2341 cccaggaaca tgcttgggca gcaggctgtg gctctgattg gctttctggc cgtcaggaac
     2401 atgtcccaac atgttgagct ctggcataga agaggctggt ggctattttg tccttgggct
     2461 gcctgttttc aggtgaggaa ggggatggta ggagacagga gacctctaaa gaccccaggt
     2521 aaaccttagc ctgttactct gaacagggta tgtgatctgc cagcagatcc ttgcgacagg
     2581 gctgggatct gatgcatgtg tgcttgtgtg agtgtgtgct gggagtcaga ttctgtgtgt
     2641 gacttttaac agcctgctcc cttgcctttt tcagggcaga agtcctccct tagagtgtgt
     2701 ctgggtacac attcaagtgc atggttgcaa actttttttt ttaaagcact gaatagtact
     2761 agacacttag taggtactta agaaatattg aatgtcgtgg tggtggtgag ctagaagtta
     2821 taaaaaaaat tctttcccaa aaacaacaac aaaaagaatt atttcattgt gaagctcagt
     2881 accacaaaaa tttaaataat tcattacaag cctttattaa aaaaaatttt ctccccaaag
     2941 taaacagaca gacaatgtct agtctatttg aaatgcctga aagcagaggg gcttcaaggc
     3001 agtgggagaa ggtgcctgtc ctctgctgga catttgacaa ccagcccttt ggatggtttg
     3061 gatgtatagg agcgaaggtg cagacagcag tggggcttag agtggggtcc tgaggctgtg
     3121 ccgtggcctt tctggggttt agccacaatc ctggcctgac tccagggcga ggcaggccaa
     3181 gggggtctgc tactgtgtcc tcccacccct acctgggctc ccatccccac agcagaggag
     3241 aaagaagcct gtcctccccg aggtcagctg cgttagagga agaagactgg gcatgtctgg
     3301 gcagagattt ccagactctg agcagcctga gatgtcagta attgtagctc gagtacgtag
     3361 taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc gtcagatcgc
     3421 ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct
     3481 ccgcgcggcc gcgccatggt gagcaagggc gaggcagtga tcaaggagtt catgcggttc
     3541 aaggtgcaca tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag
     3601 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     3661 cccttctcct gggacatcct gtcccctcag ttcatgtacg gctccagggc cttcatcaag
     3721 caccccgccg acatccccga ctactataag cagtccttcc ccgagggctt caagtgggag
     3781 cgcgtgatga acttcgagga cggcggcgcc gtgaccgtga cccaggacac ctccctggag
     3841 gacggcaccc tgatctacaa ggtgaagctc cgcggcacca acttccctcc tgacggcccc
     3901 gtaatgcaga agaagacaat gggctgggaa gcgtccaccg agcggttgta ccccgaggac
     3961 ggcgtgctga agggcgacat taagatggcc ctgcgcctga aggacggagg ccgctacctg
     4021 gcggacttca agaccaccta caaggccaag aagcccgtgc agatgcccgg cgcctacaac
     4081 gttgaccgca agttggacat cacctcccac aacgaggact acaccgtggt ggaacagtac
     4141 gaacgctccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gccaaagaaa
     4201 aagagaaaag tagatccaaa aaagaagaga aaggtagatc caaaaaagaa gagaaaggta
     4261 taggtcgaca tttatttatt tatttattta agatctctgt gccttctagt tgccagccat
     4321 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     4381 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     4441 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     4501 gggatgcggt gggctctatg gctgcagccc aagcttggcg taatcatggt catagctgtt
     4561 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4621 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4681 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc ttaaaagttt tgttacttta
     4741 tagaagaaat tttgagtttt tgtttttttt taataaataa ataaacataa ataaattgtt
     4801 tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat ctattcaaat
     4861 taataaataa acctcgatat acagaccgat aaaacacatg cgtcaatttt acgcatgatt
     4921 atctttaacg tacgtcacaa tatgattatc tttctagggt taatctagta tacgcgttta
     4981 attaatccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
     5041 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5101 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5161 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5221 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5281 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5341 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5401 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5461 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5521 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5581 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5641 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5701 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5761 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5821 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5881 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5941 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6001 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6061 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6121 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6181 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6241 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6301 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6361 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6421 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6481 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6541 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6601 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6661 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6721 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6781 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6841 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6901 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6961 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7021 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7081 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7141 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
