LOCUS       sequence-431039-        7694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of chemigenetic PKA biosensor (medium basal brightness, 
            high dynamic range) and PM targeted EGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7694)
  AUTHORS   Frei MS, Sanchez SA, He X, Liu L, Schneider F, Wang Z, Hakozaki H, 
            Li Y, Lyons AC, Rohm TV, Olefsky JM, Shi L, Schoneberg J, Fraser SE,
            Mehta S, Wang Y, Zhang J
  TITLE     Far-red chemigenetic kinase biosensors enable multiplexed and 
            super-resolved imaging of signaling networks.
  JOURNAL   Nat Biotechnol. 2025 Apr 21. doi: 10.1038/s41587-025-02642-8.
  PUBMED    40258957
REFERENCE   2  (bases 1 to 7694)
  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"; journalName: "Nat 
            Biotechnol. 2025 Apr 21. doi: 10.1038/s41587-025-02642-8."
COMMENT     Sequence Label: pcDNA3.1-HaloAKAR2.1-NES-T2A-EGFP-CAAX
FEATURES             Location/Qualifiers
     source          1..7694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2363..2389
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             2405..2458
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(2459..2461,2462..3172)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2459..2461,2462..3172)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2501..2522)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2762..2781)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3109..3130
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3285..3302)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3291..3515
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3561..3989
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3648..3667)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3858..3879
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3998..4018)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4003..4332
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4183..4318
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4245..4264
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4399..5193
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSKRIAFYRLLDEFF"
     primer_bind     complement(4453..4472)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5063..5082
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5367..5488
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5404..5423)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5458..5477
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5537..5553)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5537..5553)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5550..5572)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5561..5577
                     /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(5585..5591,5592..5609,5610..5615))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5630..5651
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5768..5785)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5939..6527)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6019..6038)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6698..7489,7490..7558))
                     /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     7321..7340
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7559..7663)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcgg ccgccaccgg atccatgctg cgtcgcgcca ccctggtgga
      961 cggcggcacc ggcggcagcg agctcagcat gcggcgggag accttccagg ccttccgcac
     1021 caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc
     1081 gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct
     1141 gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga
     1201 gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt
     1261 cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg
     1321 cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct
     1381 gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt cgacgctcga
     1441 gatttccggc gagccaacca ctggaggcag cggaggcaca ggaggcagcg gaggcacagg
     1501 aggcagcatg gcagaaatcg gtactggctt tccattcgac ccccattatg tggaagtcct
     1561 gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat ggcacccctg tgctgttcct
     1621 gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg ttgcaccgac
     1681 ccatcgctgc attgctccag acctgatcgg tatgggcaaa tccgacaaac cagacctggg
     1741 ttatttcttc gacgaccacg tccgcttcat ggatgccttc atcgaagccc tgggtctgga
     1801 agaggtcgtc ctggtcattc acgactgggg ctccgctctg ggtttccact gggccaagcg
     1861 caatccagag cgcgtcaaag gtattgcatt tatggagttc atccgcccta tcccgacctg
     1921 ggacgaatgg accatcagca agttttctca agaacagatc ggcgaaaaca ttgtgtgcag
     1981 ggtcatttgt accacgggtc aaattcccat ccgagatttg tcagctgata tttcacaagt
     2041 gcttaaggaa aaacgatcca taaagaaagt ttggacattt ggtagaaacc cagcctgtga
     2101 ctatcattta ggaaacattt caagactgtc aaataagcat ttccaaatac tactaggaga
     2161 agacggtaac cttttattga atgacatttc cactaatggg acctggttaa atgggcaaaa
     2221 agtcgagaag aacagcaatc agttactgtc tcaaggtgat gaaataaccg ttggtgtagg
     2281 cgtggaatca gatattttat ctctggtcat tttcataaac gacaaattta agcagtgcct
     2341 cgagcagaac aaagttgatc gcctgccccc cctggagcgc ctgaccctgg gccgcggcag
     2401 tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccagt
     2461 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2521 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2581 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2641 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     2701 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2761 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2821 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2881 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     2941 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     3001 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     3061 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3121 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agaagaagaa
     3181 gaagaagagc aagaccaagt gcgtgatcat gtaagaattc tgcagatatc cagcacagtg
     3241 gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     3301 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3361 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3421 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3481 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3541 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3601 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3661 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3721 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3781 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3841 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3901 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3961 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     4021 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4081 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4141 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     4201 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4261 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4321 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     4381 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4441 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     4501 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4561 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     4621 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4681 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4741 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4801 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4861 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4921 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4981 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     5041 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     5101 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gaagcgcatc
     5161 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     5221 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     5281 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5341 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     5401 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5461 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     5521 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5581 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5641 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5701 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5761 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5821 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5881 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5941 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6001 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6061 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6121 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6181 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6241 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6301 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6361 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6421 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6481 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6541 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6601 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6661 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6721 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6781 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6841 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6901 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6961 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     7021 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7081 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7141 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7201 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7261 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7321 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7381 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7441 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7501 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7561 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7621 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7681 tgccacctga cgtc
//
