LOCUS       sequence-307407-        6269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes negative-control substrate fragment of FRET-based 
            bimolecular PKA activity reporter (BimAKAR); cytosol targeted. Use 
            in conjunction with pcDNA3-Cerulean-FHA1-NES..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6269)
  AUTHORS   Herbst KJ, Allen MD, Zhang J
  TITLE     Luminescent kinase activity biosensors based on a versatile 
            bimolecular switch.
  JOURNAL   J Am Chem Soc. 2011 Apr 20;133(15):5676-9. doi: 10.1021/ja1117396. 
            Epub 2011 Mar 25.
  PUBMED    21438554
REFERENCE   2  (bases 1 to 6269)
  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"; doi: "10.1021/ja1117396";
            journalName: "J Am Chem Soc"; date: "2011-04-20- 20"; volume: "133";
            issue: "15"; pages: "5676-9"
COMMENT     Sequence Label: pcDNA3-PKAsub(T/A)-YPet-NES
FEATURES             Location/Qualifiers
     source          1..6269
                     /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"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /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(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1360..1379)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1777..1803
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(1822..1840)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1823..1840)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1860..1877)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1866..2090
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2136..2564
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2223..2242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2433..2454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2573..2593)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2578..2907
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2758..2893
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2820..2839
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2974..3768
                     /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(3028..3047)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3638..3657
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3942..4063
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3979..3998)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4033..4052
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4112..4128)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4112..4128)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4125..4147)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4136..4152
                     /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(4160..4166,4167..4184,4185..4190))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4205..4226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4343..4360)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4514..5102)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4594..4613)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5273..6064,6065..6133))
                     /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     5896..5915
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6134..6238)
                     /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 gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat gctgcgtcgc
     1021 gccgccctgg tggacggcgg caccggcggc agcatggtga gcaaaggcga agagctgttc
     1081 accggcgtgg tgcccatcct ggtggagctg gacggcgacg tgaacggcca caagttcagc
     1141 gtgagcggcg agggcgaggg cgacgccacc tacggcaagc tgaccctgaa gctgctgtgc
     1201 accaccggca agctgcccgt gccctggccc accctggtga ccaccctggg ctacggcgtg
     1261 cagtgcttcg cccggtaccc cgaccacatg aagcagcacg acttcttcaa gagcgccatg
     1321 cccgagggct acgtgcagga gcggaccatc ttcttcaagg acgacggcaa ctacaagacc
     1381 cgggccgagg tgaagttcga gggcgacacc ctggtgaacc ggatcgagct gaagggcatc
     1441 gacttcaagg aggacggcaa catcctgggc cacaagctgg agtacaacta caacagccac
     1501 aacgtgtaca tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgg
     1561 cacaacatcg aggacggcgg cgtgcagctg gccgaccact accagcagaa cacccccatc
     1621 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagag cgccctgttc
     1681 aaggacccca acgagaagcg ggaccacatg gtgctgctgg agttcctgac cgccgccggc
     1741 atcaccgagg gcatggacga gctgtacaag gaattcctgc cccccctgga gcgcctgacc
     1801 ctgtaatcta gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc
     1861 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     1921 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     1981 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2041 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2101 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2161 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2221 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2281 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2341 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2401 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2461 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2521 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2581 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2641 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2701 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     2761 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     2821 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     2881 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     2941 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3001 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3061 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3121 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3181 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3241 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3301 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3361 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3421 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3481 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3541 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3601 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3661 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3721 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     3781 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     3841 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     3901 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     3961 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4021 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4081 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4141 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4201 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4261 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4321 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4381 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4441 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4501 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4561 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4621 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4681 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4741 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4801 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4861 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4921 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4981 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5041 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5101 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5161 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5221 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5281 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5341 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5401 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5461 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5521 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5581 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5641 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5701 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5761 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5821 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5881 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5941 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6001 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6061 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6121 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6181 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6241 catttccccg aaaagtgcca cctgacgtc
//
