LOCUS       sequence-431028-        7649 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of chemigenetic Akt 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 7649)
  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 7649)
  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-HaloAktKAR2.2-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7649
                     /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             2399..2452
                     /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(2453..2455,2456..3169)
                     /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(2453..2455,2456..3166)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2495..2516)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2756..2775)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3103..3124
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3240..3257)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3246..3470
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3516..3944
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3603..3622)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3813..3834
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3953..3973)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3958..4287
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4138..4273
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4200..4219
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4354..5148
                     /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(4408..4427)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5018..5037
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5322..5443
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5359..5378)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5413..5432
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5492..5508)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5492..5508)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5505..5527)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5516..5532
                     /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(5540..5546,5547..5564,5565..5570))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5585..5606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5723..5740)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5894..6482)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5974..5993)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6653..7444,7445..7513))
                     /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     7276..7295
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7514..7618)
                     /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 cgtccgcgct cgtgcacctg
      961 gccggacccc aggccggagt ttggcggcac cggcggcagc 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 aagttttctc aagaacagat
     1981 cggcgaaaac attgtgtgca gggtcatttg taccacgggt caaattccca tccgagattt
     2041 gtcagctgat atttcacaag tgcttaagga aaaacgatcc ataaagaaag tttggacatt
     2101 tggtagaaac ccagcctgtg actatcattt aggaaacatt tcaagactgt caaataagca
     2161 tttccaaata ctactaggag aagacggtaa ccttttattg aatgacattt ccactaatgg
     2221 gacctggtta aatgggcaaa aagtcgagaa gaacagcaat cagttactgt ctcaaggtga
     2281 tgaaataacc gttggtgtag gcgtggaatc agatatttta tctctggtca ttttcataaa
     2341 cgacaaattt aagcagtgcc tcgagcagaa caaagttgat cgcggccgcg gcagtggaga
     2401 gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagtgagcaa
     2461 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     2521 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     2581 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     2641 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     2701 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2761 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2821 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2881 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     2941 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     3001 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     3061 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3121 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag aattctgcag
     3181 atatccagca cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc
     3241 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3301 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3361 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3421 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3481 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3541 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3601 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3661 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3721 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3781 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3841 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3901 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3961 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4021 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     4081 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4141 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4201 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     4261 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     4321 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     4381 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4441 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     4501 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     4561 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4621 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4681 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4741 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4801 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4861 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     4921 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4981 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     5041 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     5101 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     5161 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     5221 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     5281 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     5341 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5401 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     5461 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5521 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5581 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5641 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5701 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5761 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5821 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5881 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5941 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     6001 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     6061 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6121 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6181 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6241 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6301 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6361 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6421 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6481 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6541 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6601 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6661 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6721 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6781 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6841 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6901 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6961 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7021 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7081 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7141 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7201 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7261 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7321 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7381 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7441 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7501 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7561 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7621 catttccccg aaaagtgcca cctgacgtc
//
