LOCUS       sequence-431031-        7628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of chemigenetic PKA biosensor (high basal brightness, 
            medium dynamic range) and EGFP transfection marker in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7628)
  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 7628)
  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-HaloAKAR2.0-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7628
                     /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             2378..2431
                     /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(2432..2434,2435..3148)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2432..2434,2435..3145)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2474..2495)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2735..2754)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3082..3103
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3219..3236)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3225..3449
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3495..3923
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3582..3601)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3792..3813
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3932..3952)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3937..4266
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4117..4252
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4179..4198
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4333..5127
                     /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
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4387..4406)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4997..5016
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5301..5422
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5338..5357)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5392..5411
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5471..5487)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5471..5487)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5484..5506)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5495..5511
                     /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(5519..5525,5526..5543,5544..5549))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5564..5585
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5702..5719)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5873..6461)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5953..5972)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6632..7423,7424..7492))
                     /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     7255..7274
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7493..7597)
                     /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 agctcagcca gatgcgggag 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 gcggccgcgg cagtggagag ggcagaggaa gtctgctaac
     2401 atgcggtgac gtcgaggaga atcctggccc agtgagcaag ggcgaggagc tgttcaccgg
     2461 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2521 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     2581 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     2641 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2701 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2761 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2821 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2881 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     2941 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3001 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     3061 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3121 tctcggcatg gacgagctgt acaagtaaga attctgcaga tatccagcac agtggcggcc
     3181 gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     3241 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3301 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3361 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3421 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3481 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3541 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3601 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3661 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3721 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3781 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3841 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3901 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3961 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4021 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4081 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4141 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4201 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4261 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     4321 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4381 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4441 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4501 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4561 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4621 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4681 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4741 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4801 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4861 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4921 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4981 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5041 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     5101 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     5161 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5221 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5281 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5341 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5401 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5461 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5521 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5581 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5641 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5701 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5761 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5821 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5881 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5941 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6001 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6061 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6121 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6181 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6241 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6301 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6361 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6421 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6481 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6541 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6601 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6661 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6721 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6781 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6841 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6901 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6961 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7021 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7081 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7141 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7201 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7261 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7321 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7381 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7441 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7501 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7561 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7621 ctgacgtc
//
