LOCUS       sequence-307414-        5888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes negative-control substrate fragment of luminescence-based 
            bimolecular PKA activity reporter (LumAKAR); cytosol targeted. Use 
            in conjunction with pcDNA3-RLuc8C-FHA1-NES..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5888)
  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 5888)
  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)-RLuc8N-NES
FEATURES             Location/Qualifiers
     source          1..5888
                     /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"
     CDS             1396..1422
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(1441..1459)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1442..1459)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1479..1496)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1485..1709
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1755..2183
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1842..1861)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2052..2073
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2192..2212)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2197..2526
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2377..2512
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2439..2458
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2593..3387
                     /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(2647..2666)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3257..3276
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3561..3682
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3598..3617)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3652..3671
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3731..3747)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3731..3747)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3744..3766)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3755..3771
                     /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(3779..3785,3786..3803,3804..3809))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3824..3845
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3962..3979)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4133..4721)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4213..4232)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4892..5683,5684..5752))
                     /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     5515..5534
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5753..5857)
                     /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 agcgagctca tggcttccaa ggtgtacgac
     1081 cccgagcaac gcaaacgcat gatcactggg cctcagtggt gggctcgctg caagcaaatg
     1141 aacgtgctgg actccttcat caactactat gattccgaga agcacgccga gaacgccgtg
     1201 atttttctgc atggtaacgc tacctccagc tacctgtgga ggcacgtcgt gcctcacatc
     1261 gagcccgtgg ctagatgcat catccctgat ctgatcggaa tgggtaagtc cggcaagagc
     1321 gggaatggct catatcgcct cctggatcac tacaagtacc tcaccgcttg gttcgagctg
     1381 ctgaaccttg aattcctgcc ccccctggag cgcctgaccc tgtaatctag agggccctat
     1441 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     1501 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     1561 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     1621 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     1681 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     1741 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1801 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1861 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1921 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1981 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2041 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2101 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2161 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     2221 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2281 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2341 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2401 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2461 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     2521 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     2581 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     2641 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     2701 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     2761 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     2821 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     2881 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     2941 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3001 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3061 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3121 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3181 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     3241 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3301 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3361 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     3421 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3481 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     3541 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     3601 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     3661 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     3721 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3781 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3841 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3901 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3961 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4021 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4081 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4141 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4201 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4261 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4321 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4381 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4441 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4501 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4561 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4621 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4681 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4741 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4801 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4861 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4921 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4981 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5041 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5101 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5161 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5221 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5281 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5341 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5401 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5461 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5521 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5581 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5641 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5701 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5761 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5821 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5881 ctgacgtc
//
