LOCUS       sequence-224258-        7172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the S11A mutant of the mouse PKA catalytic 
            subunit alpha C-terminally tagged by monomeric paGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7172)
  AUTHORS   Tillo SE, Xiong WH, Takahashi M, Miao S, Andrade AL, Fortin DA, Yang
            G, Qin M, Smoody BF, Stork PJS, Zhong H
  TITLE     Liberated PKA Catalytic Subunits Associate with the Membrane via 
            Myristoylation to Preferentially Phosphorylate Membrane Substrates.
  JOURNAL   Cell Rep. 2017 Apr 18;19(3):617-629. doi: 
            10.1016/j.celrep.2017.03.070.
  PUBMED    28423323
REFERENCE   2  (bases 1 to 7172)
  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: 
            "10.1016/j.celrep.2017.03"; journalName: "Cell Rep"; date: 
            "2017-04-18- 18"; volume: "19"; issue: "3"; pages: "617-629"
COMMENT     Sequence Label: pCDNA3_PKA-C-alpha(S11A)-mPAGFP
FEATURES             Location/Qualifiers
     source          1..7172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        83..462
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        463..666
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     617..637
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     711..730
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        711..729
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1814..1840
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             join(1841..1843,1844..2554)
                     /codon_start=1
                     /product="photoactivatable GFP variant (Patterson et al., 
                     2002)"
                     /label=PA-GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1883..1904)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2144..2163)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2491..2512
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2573..2591)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2574..2591)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2611..2628)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2617..2841
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2887..3315
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2974..2993)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3184..3205
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3324..3344)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3329..3658
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3509..3644
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3571..3590
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3725..4519
                     /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(3779..3798)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4389..4408
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4693..4814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4730..4749)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4784..4803
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4863..4879)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4863..4879)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4876..4898)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4887..4903
                     /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(4911..4917,4918..4935,4936..4941))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4956..4977
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5094..5111)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5265..5853)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5345..5364)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6024..6815,6816..6884))
                     /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     6647..6666
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6885..6989)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7064..7083)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
       61 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
      121 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      181 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      241 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      301 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      361 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      421 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      481 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      541 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      601 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      661 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
      721 cactataggg agacccaagc ttgcggccgc taccatgggc aacgccgccg ccgccaagaa
      781 gggcgccgag caggagagcg tgaaagagtt cctagccaaa gccaaggaag atttcctgaa
      841 aaaatgggag accccttctc agaatacagc ccagttggat cagtttgata gaatcaagac
      901 ccttggcacc ggctcctttg ggcgagtgat gctggtgaag cacaaggaga gtgggaacca
      961 ctacgccatg aagatcttag acaagcagaa ggtggtgaag ctaaagcaga tcgagcacac
     1021 tctgaatgag aagcgcatcc tgcaggccgt caacttcccg ttcctggtca aacttgaatt
     1081 ctccttcaag gacaactcaa acctgtacat ggtcatggag tatgtagctg gtggcgagat
     1141 gttctcccac ctacggcgga ttggaaggtt cagcgagccc catgcccgtt tctacgcggc
     1201 gcagatcgtc ctgacctttg agtatctgca ctccctggac ctcatctacc gggacctgaa
     1261 gcccgagaat cttctcatcg accagcaggg ctatattcag gtgacagact tcggttttgc
     1321 caagcgtgtg aaaggccgta cttggacctt gtgtgggacc cctgagtact tggcccccga
     1381 gattatcctg agcaaaggct acaacaaggc tgtggactgg tgggctctcg gagtcctcat
     1441 ctacgagatg gctgctggtt acccaccctt cttcgctgac cagcctatcc agatctatga
     1501 gaaaatcgtc tctgggaagg tgcggttccc atcccacttc agctctgact tgaaggacct
     1561 gctgcggaac cttctgcaag tggatctcac caagcgcttt gggaacctca agaacggggt
     1621 caatgacatc aagaaccaca agtggtttgc cacgactgac tggattgcca tctatcagag
     1681 aaaggtggaa gctcccttca taccaaagtt taaaggccct ggggacacga gtaactttga
     1741 cgactatgag gaggaagaga tccgggtctc catcaatgag aagtgtggca aggagtttac
     1801 tgagtttatc gattatgatg ttcctgatta tgcaagctta atgagcaagg gcgaggagct
     1861 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1921 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1981 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcagctacgg
     2041 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2101 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2161 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2221 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2281 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggcca acttcaagat
     2341 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     2401 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagccacc agtccaagct
     2461 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2521 cgggatcact ctcggcatgg acgagctgta caagtaatct agagggccct attctatagt
     2581 gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     2641 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2701 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2761 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     2821 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     2881 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2941 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3001 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3061 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3121 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3181 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3241 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3301 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3361 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3421 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3481 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3541 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     3601 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3661 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     3721 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3781 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3841 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3901 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3961 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4021 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4081 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4141 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4201 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     4261 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4321 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4381 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4441 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4501 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     4561 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     4621 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     4681 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4741 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4801 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     4861 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4921 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4981 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5041 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5101 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5161 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5221 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5281 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5341 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5401 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5461 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5521 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5581 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5641 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5701 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5761 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5821 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5881 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5941 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6001 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6061 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6121 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6181 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6241 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6301 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6361 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6421 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6481 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6541 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6601 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6661 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6721 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6781 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6841 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6901 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6961 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     7021 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     7081 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     7141 cgagcaaaat ttaagctaca acaaggcaag gc
//
