LOCUS       sequence-224262-        7172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the S11D 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(S11D)-mPAGFP
FEATURES             Location/Qualifiers
     source          1..7172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        188..567
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        568..771
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     722..742
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     816..835
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        816..834
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1919..1945
                     /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(1946..1948,1949..2659)
                     /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(1988..2009)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2249..2268)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2596..2617
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2678..2696)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2679..2696)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2716..2733)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2722..2946
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2992..3420
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3079..3098)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3289..3310
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3429..3449)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3434..3763
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3614..3749
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3676..3695
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3830..4624
                     /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(3884..3903)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4494..4513
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4798..4919
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4835..4854)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4889..4908
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4968..4984)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4968..4984)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4981..5003)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4992..5008
                     /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(5016..5022,5023..5040,5041..5046))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5061..5082
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5199..5216)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5370..5958)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5450..5469)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6129..6920,6921..6989))
                     /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     6752..6771
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6990..7094)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(7169..7172,1..16))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
       61 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
      121 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
      181 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
      241 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      301 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      361 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      421 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      481 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      541 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      601 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      661 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      721 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta
      781 actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc
      841 caagcttgcg gccgctacca tgggcaacgc cgccgccgcc aagaagggcg atgagcagga
      901 gagcgtgaaa gagttcctag ccaaagccaa ggaagatttc ctgaaaaaat gggagacccc
      961 ttctcagaat acagcccagt tggatcagtt tgatagaatc aagacccttg gcaccggctc
     1021 ctttgggcga gtgatgctgg tgaagcacaa ggagagtggg aaccactacg ccatgaagat
     1081 cttagacaag cagaaggtgg tgaagctaaa gcagatcgag cacactctga atgagaagcg
     1141 catcctgcag gccgtcaact tcccgttcct ggtcaaactt gaattctcct tcaaggacaa
     1201 ctcaaacctg tacatggtca tggagtatgt agctggtggc gagatgttct cccacctacg
     1261 gcggattgga aggttcagcg agccccatgc ccgtttctac gcggcgcaga tcgtcctgac
     1321 ctttgagtat ctgcactccc tggacctcat ctaccgggac ctgaagcccg agaatcttct
     1381 catcgaccag cagggctata ttcaggtgac agacttcggt tttgccaagc gtgtgaaagg
     1441 ccgtacttgg accttgtgtg ggacccctga gtacttggcc cccgagatta tcctgagcaa
     1501 aggctacaac aaggctgtgg actggtgggc tctcggagtc ctcatctacg agatggctgc
     1561 tggttaccca cccttcttcg ctgaccagcc tatccagatc tatgagaaaa tcgtctctgg
     1621 gaaggtgcgg ttcccatccc acttcagctc tgacttgaag gacctgctgc ggaaccttct
     1681 gcaagtggat ctcaccaagc gctttgggaa cctcaagaac ggggtcaatg acatcaagaa
     1741 ccacaagtgg tttgccacga ctgactggat tgccatctat cagagaaagg tggaagctcc
     1801 cttcatacca aagtttaaag gccctgggga cacgagtaac tttgacgact atgaggagga
     1861 agagatccgg gtctccatca atgagaagtg tggcaaggag tttactgagt ttatcgatta
     1921 tgatgttcct gattatgcaa gcttaatgag caagggcgag gagctgttca ccggggtggt
     1981 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2041 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     2101 gctgcccgtg ccctggccca ccctcgtgac caccttcagc tacggcgtgc agtgcttcag
     2161 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2221 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2281 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2341 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2401 catggccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga
     2461 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2521 cgtgctgctg cccgacaacc actacctgag ccaccagtcc aagctgagca aagaccccaa
     2581 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2641 catggacgag ctgtacaagt aatctagagg gccctattct atagtgtcac ctaaatgcta
     2701 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2761 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2821 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2881 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     2941 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3001 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3061 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3121 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3181 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3241 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3301 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3361 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3421 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3481 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3541 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3601 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3661 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     3721 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     3781 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3841 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3901 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     3961 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4021 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4081 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4141 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4201 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4261 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4321 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4381 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4441 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4501 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4561 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4621 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4681 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4741 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     4801 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4861 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4921 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     4981 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5041 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5101 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5161 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5221 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5281 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5341 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5401 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5461 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5521 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5581 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5641 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5701 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5761 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5821 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5881 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5941 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6001 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6061 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6121 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6181 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6241 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6301 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6361 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6421 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6481 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6541 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6601 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6661 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6721 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6781 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6841 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6901 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6961 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7021 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7081 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     7141 ctcccgatcc cctatggtgc actctcagta ca
//
