LOCUS       sequence-306919-        7475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Green-fluorescent homoFRET/anisotropy-based biosensor for monitoring
            Protein Kinase A activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7475)
  AUTHORS   Ross BL, Tenner B, Markwardt ML, Zviman A, Shi G, Kerr JP, Snell NE,
            McFarland JJ, Mauban JR, Ward CW, Rizzo MA, Zhang J
  TITLE     Single-color, ratiometric biosensors for detecting signaling 
            activities in live cells.
  JOURNAL   eLife. 2018 Jul 3;7. pii: 35458. doi: 10.7554/eLife.35458
  PUBMED    29968564
REFERENCE   2  (bases 1 to 7475)
  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: "eLife."; 
            date: "2018-07-3"; volume: "7. pii: 35458. doi"; pages: " 
            10.7554/eLife.3545"
COMMENT     Sequence Label: pcDNA3-GFP-GFP-FLARE-AKAR
FEATURES             Location/Qualifiers
     source          1..7475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(49..68)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        240..619
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        620..823
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     774..794
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     868..887
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        868..886
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      906..915
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             927..944
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             948..980
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(984..992,993..1007)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(1014..1016,1017..1019,1020..1730)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2253..2255,2256..2258,2259..2972)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(3033..3051)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3034..3051)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3071..3088)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3077..3301
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3347..3775
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3434..3453)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3644..3665
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3784..3804)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3789..4118
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3969..4104
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4031..4050
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4185..4979
                     /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(4239..4258)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4849..4868
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5153..5274
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5190..5209)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5244..5263
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5323..5339)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5323..5339)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5336..5358)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5347..5363
                     /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(5371..5377,5378..5395,5396..5401))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5416..5437
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5554..5571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5725..6313)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5805..5824)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6484..7275,7276..7344))
                     /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     7107..7126
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7345..7449)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
       61 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
      121 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
      181 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      241 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      301 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      361 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      421 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      481 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      541 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      601 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      661 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      721 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      781 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      841 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagcttg
      901 cggccgccac catggtgcgg ggttctcatc atcatcatca tcatggtatg gctagcatga
      961 ctggtggaca gcaaatgggt cgggatctgt acgacgatga cgataaggat cccatggtga
     1021 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1081 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1141 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1201 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     1261 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1321 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1381 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1441 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     1501 aggccaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     1561 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1621 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1681 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag cgcatgcata
     1741 agttttctca agaacagatc ggcgaaaaca ttgtgtgcag ggtcatttgt accacgggtc
     1801 aaattcccat ccgagatttg tcagctgata tttcacaagt gcttaaggaa aaacgatcca
     1861 taaagaaagt ttggacattt ggtagaaacc cagcctgtga ctatcattta ggaaacattt
     1921 caagactgtc aaataagcat ttccaaatac tactaggaga agacggtaac cttttattga
     1981 atgacatttc cactaatggg acctggttaa atgggcaaaa agtcgagaag aacagcaatc
     2041 agttactgtc tcaaggtgat gaaataaccg ttggtgtagg cgtggaatca gatattttat
     2101 ctctggtcat tttcataaac gacaaattta agcagtgcct cgagcagaac aaagttgatc
     2161 gctctgcagg taagccaggc agcggcgagg gcagcaccaa gggcctgcgt cgcgccaccc
     2221 tggtggacgg cggcaccggc ggcagcgagc tcatggtgag caagggcgag gagctgttca
     2281 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     2341 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     2401 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     2461 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2521 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     2581 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2641 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2701 acgtctatat catggccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc
     2761 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2821 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     2881 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2941 tcactctcgg catggacgag ctgtacaagt aagaattctg cagatatcca tcacactggc
     3001 ggccgctcga gcatgcatct agagggccct attctatagt gtcacctaaa tgctagagct
     3061 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3121 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3181 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3241 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3301 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3361 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3421 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3481 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3541 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3601 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3661 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3721 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3781 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3841 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3901 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3961 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     4021 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     4081 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     4141 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4201 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4261 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4321 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4381 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4441 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4501 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4561 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4621 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4681 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4741 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4801 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4861 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4921 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4981 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     5041 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5101 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     5161 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5221 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5281 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5341 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5401 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5461 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5521 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5581 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5641 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5701 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5761 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5821 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5881 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5941 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6001 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6061 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6121 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6181 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6241 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6301 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6361 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6421 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6481 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6541 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6601 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6661 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6721 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6781 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6841 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6901 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6961 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7021 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7081 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7141 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7201 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7261 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7321 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7381 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7441 ggttccgcgc acatttcccc gaaaagtgcc acctg
//
