LOCUS       sequence-398119-        7737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses pnRFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7737)
  AUTHORS   Pang Y, Huang M, Fan Y, Yeh HW, Xiong Y, Ng HL, Ai HW
  TITLE     Development, Characterization, and Structural Analysis of a 
            Genetically Encoded Red Fluorescent Peroxynitrite Biosensor.
  JOURNAL   ACS Chem Biol. 2023 Jun 16;18(6):1388-1397. doi: 
            10.1021/acschembio.3c00139. Epub 2023 May 15.
  PUBMED    37185019
REFERENCE   2  (bases 1 to 7737)
  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: 
            "10.1021/acschembio.3c00139"; journalName: "ACS Chem Biol"; date: 
            "2023-06-16- 16"; volume: "18"; issue: "6"; pages: "1388-1397"
COMMENT     Sequence Label: pMAH-pnRFP
FEATURES             Location/Qualifiers
     source          1..7737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     505..529
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        739..757
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     739..756
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     770..789
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      822..831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1545..1564
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1619..1636)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1625..1849
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1895..2323
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1982..2001)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2192..2213
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2332..2352)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2337..2666
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2517..2652
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2579..2598
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2715..3740
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2715..3740
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    3870..3991
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3907..3926)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3961..3980
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4040..4056)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4040..4056)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4053..4075)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4064..4080
                     /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(4088..4094,4095..4112,4113..4118))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4133..4154
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4271..4288)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4442..5030)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4522..4541)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5201..5992,5993..6061))
                     /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     5824..5843
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6062..6166)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(6290..6380)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     promoter        complement(6492..6740)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(7029..7119)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     promoter        complement(7231..7479)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg
      541 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg
      601 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag
      661 agtctatagg cccaccccct tggcttcgtt agaacgcggc tacaattaat acataacctt
      721 atgtatcata cacatacgat ttaggtgaca ctatagaata acatccactt tgcctttctc
      781 tccacaggtg tccactccca ggtccaactg cacggaagct tgccaccatg gataggtcgg
      841 ctgggctcac ccgtagtttc cgagcggatg taccccgagg acggcgccct gaagagcgag
      901 atcaagtagg ggctgaggct gaaggacggc ggccactacg ccgccgaggt caagaccacc
      961 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca tcgtcgacat caagttggac
     1021 atcgtgtccc acaacgagga ctacaccatc gtggaacagt gcgaacgcgc cgagggccgc
     1081 cactccactg gcggcatgga cgagctgtac aagggaggta caggcgggag tctggtgagc
     1141 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
     1201 gagggctccg tgaacggcca caagttcgag atcgagggcg agggcgaggg ccgcccctac
     1261 gaggcctttc agaccgctaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     1321 gacatcctgt cccctcagtt catgtacggc tccaaggcct acattaagca cccagccgac
     1381 atccccgact acttcaagct gtccttcccc gagggcttca ggtgggagcg cgtggtgaac
     1441 ttcgaggacg gcggcattat tcacgttaac caggactcct ccctgcagga cggcgtattc
     1501 atctacaagg tgaagctgcg cggcaccaac ttcccccccg acggccccgt aatgcagaag
     1561 aagaccatgg gctgggaggc tacgcgtgac caagggcccg tttaaacccg ctgatcagcc
     1621 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     1681 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     1741 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     1801 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     1861 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     1921 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1981 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2041 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2101 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2161 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2221 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2281 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     2341 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2401 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     2461 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     2521 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     2581 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     2641 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     2701 ctgatcagca cgtgatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     2761 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     2821 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     2881 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     2941 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     3001 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     3061 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     3121 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     3181 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     3241 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     3301 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     3361 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     3421 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     3481 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     3541 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     3601 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     3661 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     3721 gtccgagggc aaaggaatag cacgtgctac gagatttcga ttccaccgcc gccttctatg
     3781 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3841 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     3901 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3961 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4021 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4081 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4141 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4201 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4261 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4321 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4381 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4441 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4501 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4561 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4621 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4681 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4741 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4801 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4861 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4921 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4981 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5041 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5101 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5161 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5221 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5281 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5341 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5401 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5461 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5521 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5581 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5641 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5701 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5761 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5821 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5881 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5941 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6001 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6061 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6121 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgac
     6181 tcgactcgct agtgttccaa aaaatggtgg gggacggatt cgaaccgccg aacccaaagg
     6241 gagcggattt agagtccgcg gcgtttggcc acttcgctac cccaccagag tggtctcata
     6301 cagaacttat aagattccca aatccaaaga catttcacgt ttatggtgat ttcccagaac
     6361 acatagcgac atgcaaatat gaattgatga ctgcctgatg gagaaaaaat ggtgggggac
     6421 ggattcgaac cgccgaaccc aaagggagcg gatttagagt ccgcggcgtt tggccacttc
     6481 gctaccccac cggtgtttcg tcctttccac aagatatata aagccaagaa atcgaaatac
     6541 tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa aactgcaaac
     6601 tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca
     6661 gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga
     6721 atcatgggaa ataggccctc ttcctgcccg accttcgcga tgtacgggcc agatatacgc
     6781 caatattggc cattagccat attattcatt ggttatatag cataaatcaa tattggctat
     6841 tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg ctcatgtcca
     6901 atatgaccgc catgttggct cgactcgcta gtgttccaaa aaatggtggg ggacggattc
     6961 gaaccgccga acccaaaggg agcggattta gagtccgcgg cgtttggcca cttcgctacc
     7021 ccaccagagt ggtctcatac agaacttata agattcccaa atccaaagac atttcacgtt
     7081 tatggtgatt tcccagaaca catagcgaca tgcaaatatg aattgatgac tgcctgatgg
     7141 agaaaaaatg gtgggggacg gattcgaacc gccgaaccca aagggagcgg atttagagtc
     7201 cgcggcgttt ggccacttcg ctaccccacc ggtgtttcgt cctttccaca agatatataa
     7261 agccaagaaa tcgaaatact ttcaagttac ggtaagcata tgatagtcca ttttaaaaca
     7321 taattttaaa actgcaaact acccaagaaa ttattacttt ctacgtcacg tattttgtac
     7381 taatatcttt gtgtttacag tcaaattaat tctaattatc tctctaacag ccttgtatcg
     7441 tatatgcaaa tatgaaggaa tcatgggaaa taggccctct tcctgcccga ccttcgcgat
     7501 gtacgggcca gatatacgcc aatattggcc attagccata ttattcattg gttatatagc
     7561 ataaatcaat attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat
     7621 ttatattggc tcatgtccaa tatgaccgcc atgttggctc gagcattgat tattgactag
     7681 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccg
//
