LOCUS       sequence-224241-        7163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the G2A 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 7163)
  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 7163)
  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_nmPKA-C-alpha-mPAGFP
FEATURES             Location/Qualifiers
     source          1..7163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        103..482
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        483..686
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     637..657
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     731..750
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        731..749
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1825..1851
                     /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(1852..1854,1855..2565)
                     /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(1894..1915)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2155..2174)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2502..2523
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2584..2602)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2585..2602)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2622..2639)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2628..2852
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2898..3326
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2985..3004)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3195..3216
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3335..3355)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3340..3669
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3520..3655
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3582..3601
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3736..4530
                     /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(3790..3809)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4400..4419
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4704..4825
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4741..4760)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4795..4814
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4874..4890)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4874..4890)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4887..4909)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4898..4914
                     /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(4922..4928,4929..4946,4947..4952))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4967..4988
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5105..5122)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5276..5864)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5356..5375)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6035..6826,6827..6895))
                     /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     6658..6677
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6896..7000)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7075..7094)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
       61 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
      121 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      181 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      241 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      301 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      361 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      421 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      481 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      541 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      601 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      661 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc
      721 ttatcgaaat taatacgact cactataggg agacccaagc ttaccatggc caacgccgcc
      781 gccgccaaga agggcagcga gcaggagagc gtgaaagagt tcctagccaa agccaaggaa
      841 gatttcctga aaaaatggga gaccccttct cagaatacag cccagttgga tcagtttgat
      901 agaatcaaga cccttggcac cggctccttt gggcgagtga tgctggtgaa gcacaaggag
      961 agtgggaacc actacgccat gaagatctta gacaagcaga aggtggtgaa gctaaagcag
     1021 atcgagcaca ctctgaatga gaagcgcatc ctgcaggccg tcaacttccc gttcctggtc
     1081 aaacttgaat tctccttcaa ggacaactca aacctgtaca tggtcatgga gtatgtagct
     1141 ggtggcgaga tgttctccca cctacggcgg attggaaggt tcagcgagcc ccatgcccgt
     1201 ttctacgcgg cgcagatcgt cctgaccttt gagtatctgc actccctgga cctcatctac
     1261 cgggacctga agcccgagaa tcttctcatc gaccagcagg gctatattca ggtgacagac
     1321 ttcggttttg ccaagcgtgt gaaaggccgt acttggacct tgtgtgggac ccctgagtac
     1381 ttggcccccg agattatcct gagcaaaggc tacaacaagg ctgtggactg gtgggctctc
     1441 ggagtcctca tctacgagat ggctgctggt tacccaccct tcttcgctga ccagcctatc
     1501 cagatctatg agaaaatcgt ctctgggaag gtgcggttcc catcccactt cagctctgac
     1561 ttgaaggacc tgctgcggaa ccttctgcaa gtggatctca ccaagcgctt tgggaacctc
     1621 aagaacgggg tcaatgacat caagaaccac aagtggtttg ccacgactga ctggattgcc
     1681 atctatcaga gaaaggtgga agctcccttc ataccaaagt ttaaaggccc tggggacacg
     1741 agtaactttg acgactatga ggaggaagag atccgggtct ccatcaatga gaagtgtggc
     1801 aaggagttta ctgagtttat cgattatgat gttcctgatt atgcaagctt aatgagcaag
     1861 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1921 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1981 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2041 ttcagctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     2101 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2161 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2221 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2281 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcc
     2341 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2401 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagccac
     2461 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2521 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaatc tagagggccc
     2581 tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag
     2641 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2701 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2761 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2821 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2881 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2941 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3001 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3061 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3121 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3181 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3241 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3301 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     3361 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3421 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3481 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3541 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3601 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3661 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     3721 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3781 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3841 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3901 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3961 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     4021 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     4081 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     4141 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     4201 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     4261 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4321 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4381 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4441 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4501 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4561 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4621 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4681 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4741 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4801 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4861 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4921 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4981 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5041 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5101 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5161 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5221 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5281 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5341 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5401 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5461 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5521 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5581 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5641 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5701 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5761 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5821 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5881 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5941 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6001 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6061 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6121 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6181 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6241 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6301 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6361 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6421 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6481 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6541 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6601 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6661 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6721 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6781 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6841 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6901 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6961 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7021 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     7081 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     7141 tgagtagtgc gcgagcaaaa ttt
//
