LOCUS       sequence-224234-        6251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the G2A mutant of the N-terminal 47 residues
            of 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 6251)
  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 6251)
  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-N-mPAGFP
FEATURES             Location/Qualifiers
     source          1..6251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        164..543
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        544..747
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     698..718
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     792..811
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        792..810
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             974..1000
                     /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(1001..1003,1004..1714)
                     /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(1043..1064)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1304..1323)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1651..1672
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1733..1751)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1734..1751)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1771..1788)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1777..2001
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2047..2475
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2134..2153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2344..2365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2484..2504)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2489..2818
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2669..2804
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2731..2750
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2885..3679
                     /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(2939..2958)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3549..3568
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3853..3974
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3890..3909)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3944..3963
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4023..4039)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4023..4039)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4036..4058)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4047..4063
                     /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(4071..4077,4078..4095,4096..4101))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4116..4137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4254..4271)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4425..5013)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4505..4524)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5184..5975,5976..6044))
                     /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     5807..5826
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6045..6149)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6224..6243)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
       61 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
      121 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
      181 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      241 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      301 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      361 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      421 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      481 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      541 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      601 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      661 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      721 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
      781 cttatcgaaa ttaatacgac tcactatagg gagacccaag cttaccatgg ccaacgccgc
      841 cgccgccaag aagggcagcg agcaggagag cgtgaaagag ttcctagcca aagccaagga
      901 agatttcctg aaaaaatggg agaccccttc tcagaataca gcccagttgg atcagtttga
      961 tagaatcatc gattatgatg ttcctgatta tgcaagctta atgagcaagg gcgaggagct
     1021 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1081 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1141 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcagctacgg
     1201 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1261 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1321 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1381 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1441 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggcca acttcaagat
     1501 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1561 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagccacc agtccaagct
     1621 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1681 cgggatcact ctcggcatgg acgagctgta caagtaatct agagggccct attctatagt
     1741 gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     1801 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     1861 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     1921 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     1981 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     2041 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2101 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2161 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2221 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2281 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2341 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2401 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2461 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2521 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2581 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2641 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2701 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     2761 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     2821 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     2881 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     2941 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3001 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3061 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3121 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3181 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3241 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3301 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3361 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     3421 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     3481 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3541 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     3601 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     3661 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     3721 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     3781 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     3841 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3901 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3961 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     4021 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4081 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4141 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4201 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4261 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4321 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4381 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4441 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4501 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4561 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4621 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4681 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4741 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4801 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4861 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4921 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4981 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5041 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5101 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5161 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5221 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5281 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5341 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5401 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5461 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5521 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5581 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5641 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5701 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5761 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5821 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5881 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5941 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6001 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6061 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6121 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6181 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     6241 ccgcatagtt a
//
