LOCUS       sequence-431035-        7367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of chemigenetic ERK 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 7367)
  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 7367)
  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-HaloEKAR2.2-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7367
                     /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             2138..2191
                     /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(2192..2194,2195..2908)
                     /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(2192..2194,2195..2905)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2234..2255)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2495..2514)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2842..2863
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2979..2996)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2985..3209
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3255..3683
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3342..3361)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3552..3573
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3692..3712)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3857..3991
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3918..3937
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4072..4866
                     /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(4126..4145)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4736..4755
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5040..5161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5077..5096)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5131..5150
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5210..5226)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5210..5226)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5223..5245)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5234..5250
                     /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(5258..5264,5265..5282,5283..5288))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5303..5324
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5441..5458)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5612..6200)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5692..5711)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6371..7162,7163..7231))
                     /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     6994..7013
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7232..7336)
                     /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 atccatgcct gacgtaccac gtacaccagt
      961 aggaaaggca aagctgtcat tccaattccc ccgcgcacgg gagctcagct ggcggcggga
     1021 gaccttccag gccttccgca ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt
     1081 ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga
     1141 ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa
     1201 cgagctgcca atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag aatacatgga
     1261 ctggctgcac cagtcccctg tcccgaagct gctgttctgg ggcaccccag gcgttctgat
     1321 cccaccggcc gaagccgctc gcctggccaa aagcctgcct aactgcaagg ctgtggacat
     1381 cggcccgggt ctgaatctgc tgcaagaaga caacccggac ctgatcggca gcgagatcgc
     1441 gcgctggctg tcgacgctcg agatttccgg cgagccaacc actggaggca gcggaggcac
     1501 aggaggcagc ggaggcacag gaggcagcat ggcagaaatc ggtactggct ttccattcga
     1561 cccccattat gtggaagtcc tgggcgagcg catgcactac gtcgatgttg gtccgcgcga
     1621 tggcacccct gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat
     1681 catcccgcat gttgcaccga cccatcgctg cattgctcca gacctgatcg gtatgggcaa
     1741 atccgacaaa ccagacctgg gttatttctt cgacgaccac gtccgcttca tggatgcctt
     1801 catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt cacgactggg gctccgctct
     1861 gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt
     1921 catccgccct atcccgacct gggacgaatg gaccatcagc atggcggacg aggagaagct
     1981 gccgcccggc tgggagaagc gcatgagccg cagctcaggc cgagtgtact acttcaacca
     2041 catcactaac gccagccagt gggagcggcc cagcggcaac agcagcagtg gtggcaaaaa
     2101 cgggcagggg gagcctgcca ggggccgcgg cagtggagag ggcagaggaa gtctgctaac
     2161 atgcggtgac gtcgaggaga atcctggccc agtgagcaag ggcgaggagc tgttcaccgg
     2221 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2281 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     2341 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     2401 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2461 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2521 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2581 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2641 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     2701 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     2761 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     2821 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     2881 tctcggcatg gacgagctgt acaagtaaga attctgcaga tatccagcac agtggcggcc
     2941 gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     3001 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3061 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3121 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3181 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3241 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3301 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3361 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3421 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3481 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3541 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3601 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3661 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3721 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3781 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3841 tagtcagcaa ccatagtccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3901 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3961 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     4021 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     4081 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4141 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4201 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4261 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4321 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4381 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4441 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4501 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4561 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4621 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4681 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4741 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4801 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4861 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4921 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4981 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     5041 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     5101 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5161 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5221 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5281 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5341 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5401 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5461 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5521 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5581 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5641 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5701 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5761 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5821 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5881 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5941 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6001 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6061 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6121 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6181 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6241 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6301 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6361 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6421 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6481 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6541 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6601 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6661 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6721 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6781 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6841 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6901 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6961 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7021 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7081 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7141 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7201 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7261 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7321 aaataggggt tccgcgcact tttcggggaa atgtgccacc tgacgtc
//
