LOCUS       sequence-431037-        7646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of chemigenetic PKC biosensor (low basal brightness, 
            extremly high 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 7646)
  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 7646)
  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-HaloCKAR2.2-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(43..62)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        234..613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        614..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     768..788
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     862..881
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        862..880
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2395..2448
                     /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(2449..2451,2452..3165)
                     /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(2449..2451,2452..3162)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2491..2512)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2752..2771)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3099..3120
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3236..3253)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3242..3466
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3512..3940
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3599..3618)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3809..3830
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3949..3969)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3954..4283
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4134..4269
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4196..4215
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4350..5144
                     /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(4404..4423)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5014..5033
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5318..5439
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5355..5374)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5409..5428
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5488..5504)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5488..5504)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5501..5523)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5512..5528
                     /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(5536..5542,5543..5560,5561..5566))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5581..5602
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5719..5736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5890..6478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5970..5989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6649..7440,7441..7509))
                     /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     7272..7291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7510..7614)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
       61 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
      121 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
      181 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
      241 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      361 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      421 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      481 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      541 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      601 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      661 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      721 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      781 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
      841 tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg
      901 tttaaactta agcttgcggc cgccaccgga tccatgcgat tcagaagatt ccagacgttg
      961 aaggacaagg ctaaggctgg cggcaccggc ggcagcgagc tcagctggcg gcgggagacc
     1021 ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca gaacgttttt
     1081 atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat
     1141 taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt cccaaacgag
     1201 ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata catggactgg
     1261 ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt tctgatccca
     1321 ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc
     1381 ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga gatcgcgcgc
     1441 tggctgtcga cgctcgagat ttccggcgag ccaaccactg gaggcagcgg aggcacagga
     1501 ggcagcggag gcacaggagg cagcatggca gaaatcggta ctggctttcc attcgacccc
     1561 cattatgtgg aagtcctggg cgagcgcatg cactacgtcg atgttggtcc gcgcgatggc
     1621 acccctgtgc tgttcctgca cggtaacccg acctcctcct acgtgtggcg caacatcatc
     1681 ccgcatgttg caccgaccca tcgctgcatt gctccagacc tgatcggtat gggcaaatcc
     1741 gacaaaccag acctgggtta tttcttcgac gaccacgtcc gcttcatgga tgccttcatc
     1801 gaagccctgg gtctggaaga ggtcgtcctg gtcattcacg actggggctc cgctctgggt
     1861 ttccactggg ccaagcgcaa tccagagcgc gtcaaaggta ttgcatttat ggagttcatc
     1921 cgccctatcc cgacctggga cgaatggacc atcagcaagt tttctcaaga acagatcggc
     1981 gaaaacattg tgtgcagggt catttgtacc acgggtcaaa ttcccatccg agatttgtca
     2041 gctgatattt cacaagtgct taaggaaaaa cgatccataa agaaagtttg gacatttggt
     2101 agaaacccag cctgtgacta tcatttagga aacatttcaa gactgtcaaa taagcatttc
     2161 caaatactac taggagaaga cggtaacctt ttattgaatg acatttccac taatgggacc
     2221 tggttaaatg ggcaaaaagt cgagaagaac agcaatcagt tactgtctca aggtgatgaa
     2281 ataaccgttg gtgtaggcgt ggaatcagat attttatctc tggtcatttt cataaacgac
     2341 aaatttaagc agtgcctcga gcagaacaaa gttgatcgcg gccgcggcag tggagagggc
     2401 agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccagt gagcaagggc
     2461 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     2521 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     2581 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     2641 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     2701 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     2761 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2821 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     2881 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     2941 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     3001 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     3061 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     3121 accgccgccg ggatcactct cggcatggac gagctgtaca agtaagaatt ctgcagatat
     3181 ccagcacagt ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     3241 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     3301 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     3361 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     3421 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     3481 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     3541 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3601 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3661 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3721 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3781 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3841 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3901 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     3961 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4021 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4081 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     4141 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4201 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     4261 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     4321 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     4381 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     4441 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     4501 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     4561 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4621 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4681 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4741 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4801 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4861 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4921 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4981 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     5041 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     5101 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     5161 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     5221 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     5281 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     5341 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     5401 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     5461 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     5521 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     5581 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5641 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5701 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5761 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5821 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5881 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5941 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6001 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6061 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6121 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6181 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6241 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6301 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6361 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6421 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6481 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6541 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6601 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6661 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6721 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6781 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6841 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6901 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6961 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7021 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7081 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7141 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7201 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7261 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7321 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7381 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7441 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7501 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7561 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7621 tccccgaaaa gtgccacctg acgtcg
//
