LOCUS       sequence-328333-        7701 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses reversibly switchable A-kinase activity reporter with 
            EV-linker (rsAKARev) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7701)
  AUTHORS   Roebroek T, Vandenberg W, Sipieter F, Hugelier S, Stove C, Zhang J, 
            Dedecker P
  TITLE     Simultaneous readout of multiple FRET pairs using photochromism.
  JOURNAL   Nat Commun. 2021 Mar 31;12(1):2005. doi: 10.1038/s41467-021-22043-0.
  PUBMED    33790271
REFERENCE   2  (bases 1 to 7701)
  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 
            Commun."; date: "2021-03-31"; volume: "12(1):2005. doi"; pages: " 
            10.1038/s41467-021-22043-0"
COMMENT     Sequence Label: pcDNA3.1-rsAKARev
FEATURES             Location/Qualifiers
     source          1..7701
                     /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             914..1621
                     /codon_start=1
                     /product="monomeric teal (cyan) fluorescent protein (Ai et 
                     al., 2006)"
                     /label=mTFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTNAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTLKWEPSTEILYVRDGVLV
                     GDIKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESA
                     VARYSTGGMDELYK"
     CDS             2063..2410
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             2456..3190
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     primer_bind     2590..2611
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2717..2738)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2978..2997)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(3254..3272)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3255..3272)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3292..3309)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3298..3522
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3568..3996
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3655..3674)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3865..3886
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4005..4025)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4010..4339
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4190..4325
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4252..4271
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4406..5200
                     /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(4460..4479)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5070..5089
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5374..5495
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5411..5430)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5465..5484
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5544..5560)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5544..5560)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5557..5579)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5568..5584
                     /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(5592..5598,5599..5616,5617..5622))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5637..5658
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5775..5792)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5946..6534)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6026..6045)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6705..7496,7497..7565))
                     /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     7328..7347
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7566..7670)
                     /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 gcttggtacc
      901 gagctcggat cccatggtga gcaagggcga ggagaccaca atgggcgtaa tcaagcccga
      961 catgaagatc aagctgaaga tggagggcaa cgtgaatggc cacgccttcg tgatcgaggg
     1021 cgagggcgag ggcaagccct acgacggcac caacaccatc aacctggagg tgaaggaggg
     1081 agcccccctg cccttctcct acgacattct gaccaacgcc ttcgcttacg gcaacagggc
     1141 cttcaccaag taccccgacg acatccccaa ctacttcaag cagtccttcc ccgagggcta
     1201 ctcttgggag cgcaccatga ccttcgagga caagggcatc gtgaaggtga agtccgacat
     1261 ctccatggag gaggactcct tcatctacga gatacacctc aagggcgaga acttcccccc
     1321 caacggcccc gtgatgcaga agaagaccct gaagtgggag ccctccaccg agatcctgta
     1381 cgtgcgcgac ggcgtgctgg tgggcgacat caagcacaag ctgctgctgg agggcggcgg
     1441 ccaccaccgc gttgacttca agaccatcta cagggccaag aaggcggtga agctgcccga
     1501 ctaccacttt gtggaccacc gcatcgagat cctgaaccac gacaaggact acaacaaggt
     1561 gaccgtttac gagagcgccg tggcccgcta ctccaccggc ggcatggacg agctgtacaa
     1621 gagatctatg cataagtttt ctcaagaaca gatcggcgaa aacattgtgt gcagggtcat
     1681 ttgtaccacg ggtcaaattc ccatccgaga tttgtcagct gatatttcac aagtgcttaa
     1741 ggaaaaacga tccataaaga aagtttggac atttggtaga aacccagcct gtgactatca
     1801 tttaggaaac atttcaagac tgtcaaataa gcatttccaa atactactag gagaagacgg
     1861 taacctttta ttgaatgaca tttccactaa tgggacctgg ttaaatgggc aaaaagtcga
     1921 gaagaacagc aatcagttac tgtcccaagg tgatgaaata accgttggtg taggcgtgga
     1981 atcagatatt ttatctctgg tcattttcat aaacgacaaa tttaagcagt gcctggagca
     2041 gaacaaagtt gatcgcggta ccagtgctgg tggtagtgct ggtggtagtg ctggtggtag
     2101 tgctggtggt agtgctggtg gttccggcag tgctggtggt agtgctggtg gtagtaccag
     2161 tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggttc
     2221 cggcagtgct ggtggtagtg ctggtggtag taccagtgct ggtggtagtg ctggtggtag
     2281 tgctggtggt agtgctggtg gtagtgctgg tggttccggc agtgctggtg gtagtgctgg
     2341 tggtagtacc agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggtag
     2401 tgctggtggt tccggattga ggcgcgcgac gctggttgac ggcggcagcg agctcatggg
     2461 cggcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2521 gctgcccgac aaccactacc tgagctacca gtccaagctg agcaaagacc ccaacgagaa
     2581 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2641 cgagctgtac aagggaggta ccggtggatc tatggtgagc aagggcgagg agctgttcac
     2701 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     2761 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagc tgatctgcac
     2821 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgggct acggccttca
     2881 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     2941 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     3001 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     3061 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     3121 cgtctatatc accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca
     3181 caacatcgag taagaattct gcagatatcc atcacactgg cggccgctcg agcatgcatc
     3241 tagagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg
     3301 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3361 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3421 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3481 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3541 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3601 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3661 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3721 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3781 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3841 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3901 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3961 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     4021 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4081 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4141 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4201 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4261 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     4321 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     4381 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4441 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4501 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4561 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4621 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4681 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4741 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4801 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4861 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4921 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4981 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     5041 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     5101 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5161 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     5221 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     5281 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5341 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     5401 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5461 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5521 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5581 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5641 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5701 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5761 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5821 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5881 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5941 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     6001 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6061 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6121 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6181 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6241 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6301 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6361 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6421 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6481 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6541 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6601 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6661 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6721 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6781 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6841 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6901 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6961 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     7021 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     7081 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7141 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7201 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7261 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7321 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7381 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7441 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7501 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7561 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7621 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7681 cgaaaagtgc cacctgacgt c
//
