LOCUS       sequence-201711-        7042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express PKA-SPARK, an activity reporter of protein kinase A in 
            mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7042)
  AUTHORS   Zhang Q, Huang H, Lu Q, Wu R, Chung CI, Zhang SQ, Torra J, Schepis 
            A, Coughlin SR, Kornberg TB, Shu X
  TITLE     Visualizing Dynamics of Cell Signaling In Vivo with a Phase 
            Separation-Based Kinase Reporter.
  JOURNAL   Mol Cell. 2018 Jan 3. pii: S1097-2765(17)30936-X. doi: 
            10.1016/j.molcel.2017.12.008.
  PUBMED    29307513
REFERENCE   2  (bases 1 to 7042)
  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: "Mol Cell. 
            2018 Jan 3. pii: S1097-2765(17)30936-X. doi: 
            10.1016/j.molcel.2017.12.008."
COMMENT     Sequence Label: pcDNA3-PKA-SPARK
FEATURES             Location/Qualifiers
     source          1..7042
                     /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             join(997..999,1000..1002,1003..1713)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1042..1063)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1303..1322)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1650..1671
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1909..1962
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(2636..2653)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2642..2866
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2912..3340
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2999..3018)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3209..3230
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3349..3369)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3354..3683
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3534..3669
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3596..3615
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3750..4544
                     /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(3804..3823)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4414..4433
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4718..4839
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4755..4774)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4809..4828
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4888..4904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4888..4904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4901..4923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4912..4928
                     /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(4936..4942,4943..4960,4961..4966))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4981..5002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5119..5136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5290..5875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5370..5389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6046..6837,6838..6906))
                     /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     6669..6688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6907..7011)
                     /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 gccaccatgg gcggtggagg gagcctgcgt cgcgccaccc tggtggacgg cggactcgag
      961 tccggactca gatctggcag cggtggaggc gatatcatgg tgagcaaggg cgaggagctg
     1021 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1081 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     1141 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     1201 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1261 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     1321 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     1381 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     1441 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
     1501 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     1561 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     1621 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1681 gggatcactc tcggcatgga cgagctgtac aagggatccg gcagtgctgg tggtagtgct
     1741 ggtggtagtg ctggtggtag tgctggtggc agtgctggtg gtagtgctgg tggtagtgct
     1801 ggtggctcta gaggagaaat cgcaaagagt ctgaaagaaa ttgcaaaatc cttgaaggaa
     1861 atagcgtggt cactgaaaga aattgccaag tccctcaaag ggggtaccga gggcagagga
     1921 agtctgctaa catgcggtga cgtcgaggag aatcctggcc caaagcttgc caccatgcat
     1981 aagttttctc aagaacagat cggcgaaaac attgtgtgca gggtcatttg taccacgggt
     2041 caaattccca tccgagattt gtcagctgat atttcacaag tgcttaagga aaaacgatcc
     2101 ataaagaaag tttggacatt tggtagaaac ccagcctgtg actatcattt aggaaacatt
     2161 tcaagactgt caaataagca tttccaaata ctactaggag aagacggtaa ccttttattg
     2221 aatgacattt ccactaatgg gacctggtta aatgggcaaa aagtcgagaa gaacagcaat
     2281 cagttactgt ctcaaggtga tgaaataacc gttggtgtag gcgtggaatc agatatttta
     2341 tctctggtca ttttcataaa cgacaaattt aagcagtgcc tcgaacagaa caaagttgat
     2401 cgcggatccg gcagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggc
     2461 agtgctggtg gtagtgctgg tggtagtgct ggtggctcta gaaccttgag agaaatcgag
     2521 gaattgttga ggaaaatcat tgaagacagc gtgcgcagtg tggccgagct ggaggacatt
     2581 gagaagtggc tgaagaaaat ctaagaattc gggcccgttt aaacccgctg atcagcctcg
     2641 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2701 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2761 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2821 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2881 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2941 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3001 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3061 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3121 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3181 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3241 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3301 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     3361 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3421 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3481 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3541 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3601 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     3661 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     3721 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3781 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3841 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3901 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3961 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4021 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4081 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4141 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4201 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4261 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4321 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4381 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4441 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4501 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4561 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4621 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4681 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     4741 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4801 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     4861 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4921 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4981 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5041 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5101 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5161 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5221 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5281 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5341 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5401 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5461 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5521 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5581 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5641 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5701 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5761 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5821 tagcggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5881 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5941 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6001 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6061 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6121 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6181 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6241 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6301 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6361 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6421 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6481 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6541 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6601 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6661 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6721 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6781 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6841 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6901 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6961 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7021 ccgaaaagtg ccacctgacg tc
//
