LOCUS       sequence-229914-        6632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cpGFP-based excitation-ratiometric biosensor for monitoring Protein 
            Kinase A activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6632)
  AUTHORS   Mehta S, Zhang Y, Roth RH, Zhang JF, Mo A, Tenner B, Huganir RL, 
            Zhang J
  TITLE     Single-fluorophore biosensors for sensitive and multiplexed 
            detection of signalling activities.
  JOURNAL   Nat Cell Biol. 2018 Sep 24. pii: 10.1038/s41556-018-0200-6. doi: 
            10.1038/s41556-018-0200-6.
  PUBMED    30250062
REFERENCE   2  (bases 1 to 6632)
  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 Cell 
            Biol. 2018 Sep 24. pii: 10.1038/s41556-018-0200-6. doi: 
            10.1038/s41556-018-0200-6."
COMMENT     Sequence Label: pcDNA3.1(+)-ExRai-AKAR
FEATURES             Location/Qualifiers
     source          1..6632
                     /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"
     primer_bind     1201..1222
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1328..1349)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1589..1608)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2223..2240)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2229..2453
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2499..2927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2586..2605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2796..2817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2936..2956)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2941..3270
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3121..3256
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3183..3202
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3337..4131
                     /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(3391..3410)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4001..4020
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4305..4426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4342..4361)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4396..4415
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4475..4491)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4475..4491)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4488..4510)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4499..4515
                     /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(4523..4529,4530..4547,4548..4553))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4568..4589
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4706..4723)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4877..5465)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4957..4976)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5636..6427,6428..6496))
                     /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     6259..6278
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6497..6601)
                     /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 agctcagcgc gatcaacgtc tatatcaagg ccgacaagca
     1021 gaagaacggc atcaaggcga acttcaagat ccgccacaac atcgaggacg gcggcgtgca
     1081 gctcgcctac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1141 caaccactac ctgagcgtgc agtccaaact ttcgaaagac cccaacgaga agcgcgatca
     1201 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     1261 caagggcggt accggaggga gcatggtgag caagggcgag gagctgttca ccggggtggt
     1321 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1381 gggtgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1441 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1501 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1561 catccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1621 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1681 ggacggcaac atcctggggc acaagctgga gtacaacagc atgcataagt tttctcaaga
     1741 acagatcggc gaaaacattg tgtgcagggt catttgtacc acgggtcaaa ttcccatccg
     1801 agatttgtca gctgatattt cacaagtgct taaggaaaaa cgatccataa agaaagtttg
     1861 gacatttggt agaaacccag cctgtgacta tcatttagga aacatttcaa gactgtcaaa
     1921 taagcatttc caaatactac taggagaaga cggtaacctt ttattgaatg acatttccac
     1981 taatgggacc tggttaaatg ggcaaaaagt cgagaagaac agcaatcagt tactgtctca
     2041 aggtgatgaa ataaccgttg gtgtaggcgt ggaatcagat attttatctc tggtcatttt
     2101 cataaacgac aaatttaagc agtgcctcga gcagaacaaa gttgatcgct aagaattctg
     2161 cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc
     2221 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2281 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2341 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2401 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2461 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2521 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2581 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2641 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2701 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2761 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2821 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2881 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     2941 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3001 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3061 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3121 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3181 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3241 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3301 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3361 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3421 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3481 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3541 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3601 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3661 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3721 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3781 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3841 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     3901 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3961 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4021 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4081 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4141 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4201 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4261 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4321 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4381 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4441 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4501 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4561 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4621 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4681 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4741 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4801 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4861 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4921 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4981 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5041 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5101 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5161 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5221 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5281 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5341 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5401 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5461 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5521 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5581 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5641 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5701 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5761 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5821 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5881 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5941 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6001 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6061 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6121 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6181 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6241 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6301 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6361 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6421 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6481 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6541 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6601 gcacatttcc ccgaaaagtg ccacctgacg tc
//
