LOCUS       sequence-215462-        7164 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 7164)
  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 7164)
  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..7164
                     /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"
     CDS             1957..1983
                     /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(1984..1986,1987..2697)
                     /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(2026..2047)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2287..2306)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2634..2655
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2716..2734)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2717..2734)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2754..2771)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2760..2984
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3030..3458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3117..3136)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3327..3348
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3467..3487)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3472..3802
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3653..3788
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3715..3734
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3869..4663
                     /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(3923..3942)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4533..4552
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4837..4958
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4874..4893)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4928..4947
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5007..5023)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5007..5023)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5020..5042)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5031..5047
                     /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(5055..5061,5062..5079,5080..5085))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5100..5121
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5238..5255)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5409..5997)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5489..5508)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6168..6959,6960..7028))
                     /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     6791..6810
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7029..7133)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 gcttaccatg
      901 gccaacgccg ccgccgccaa gaagggcagc gagcaggaga gcgtgaaaga gttcctagcc
      961 aaagccaagg aagatttcct gaaaaaatgg gagacccctt ctcagaatac agcccagttg
     1021 gatcagtttg atagaatcaa gacccttggc accggctcct ttgggcgagt gatgctggtg
     1081 aagcacaagg agagtgggaa ccactacgcc atgaagatct tagacaagca gaaggtggtg
     1141 aagctaaagc agatcgagca cactctgaat gagaagcgca tcctgcaggc cgtcaacttc
     1201 ccgttcctgg tcaaacttga attctccttc aaggacaact caaacctgta catggtcatg
     1261 gagtatgtag ctggtggcga gatgttctcc cacctacggc ggattggaag gttcagcgag
     1321 ccccatgccc gtttctacgc ggcgcagatc gtcctgacct ttgagtatct gcactccctg
     1381 gacctcatct accgggacct gaagcccgag aatcttctca tcgaccagca gggctatatt
     1441 caggtgacag acttcggttt tgccaagcgt gtgaaaggcc gtacttggac cttgtgtggg
     1501 acccctgagt acttggcccc cgagattatc ctgagcaaag gctacaacaa ggctgtggac
     1561 tggtgggctc tcggagtcct catctacgag atggctgctg gttacccacc cttcttcgct
     1621 gaccagccta tccagatcta tgagaaaatc gtctctggga aggtgcggtt cccatcccac
     1681 ttcagctctg acttgaagga cctgctgcgg aaccttctgc aggtggatct caccaagcgc
     1741 tttgggaacc tcaagaacgg ggtcaatgac atcaagaacc acaagtggtt tgccacgact
     1801 gactggattg ccatctatca gagaaaggtg gaagctccct tcataccaaa gtttaaaggc
     1861 cctggggaca cgagtaactt tgacgactat gaggaggaag agatccgggt ctccatcaat
     1921 gagaagtgtg gcaaggagtt tactgagttt atcgattatg atgttcctga ttatgcaagc
     1981 ttaatgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2041 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2101 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2161 ctcgtgacca ccttcagcta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     2221 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2281 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2341 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2401 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2461 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2521 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2581 tacctgagcc accagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2641 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     2701 tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac
     2761 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2821 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2881 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2941 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3001 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3061 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3121 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3181 tcggggcatc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3241 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3301 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3361 ccctatctcg gtctattctt ttgatttata agggattttg gggatttcgg cctattggtt
     3421 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3481 ttagggtgtg gaaagtcccc aggctcccca ggcaggcaga agtatgcaaa gcatgcatct
     3541 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3601 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3661 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3721 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     3781 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     3841 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     3901 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     3961 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4021 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     4081 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     4141 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     4201 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     4261 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     4321 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     4381 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     4441 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     4501 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4561 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4621 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     4681 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     4741 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4801 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4861 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4921 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4981 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5041 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5101 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5161 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5221 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5281 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5341 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5401 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5461 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5521 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5581 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     5641 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5701 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5761 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5821 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     5881 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5941 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6001 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6061 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6121 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6181 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6241 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6301 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6361 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6421 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6481 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6541 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6601 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6661 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6721 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6781 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6841 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6901 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6961 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7021 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7081 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7141 ccccgaaaag tgccacctga cgtc
//
