LOCUS       sequence-224089-        7172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the mouse PKA catalytic subunit alpha 
            C-terminally tagged by monomeric mEGFP. Silent mutations were 
            introduced to make it resistant to shRNA knock-down..
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_shRNA resistant PKA-C-alpha-mEGFP
FEATURES             Location/Qualifiers
     source          1..7172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        172..551
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        552..755
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     706..726
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     800..819
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        800..818
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1903..1929
                     /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(1930..1932,1933..2646)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1972..1993)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2233..2252)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2580..2601
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2662..2680)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2663..2680)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2700..2717)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2706..2930
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2976..3404
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3063..3082)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3273..3294
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3413..3433)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3418..3747
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3598..3733
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3660..3679
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3814..4608
                     /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(3868..3887)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4478..4497
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4782..4903
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4819..4838)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4873..4892
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4952..4968)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4952..4968)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4965..4987)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4976..4992
                     /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(5000..5006,5007..5024,5025..5030))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5045..5066
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5183..5200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5354..5942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6113..6904,6905..6973))
                     /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     6736..6755
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6974..7078)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7153..7172)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
       61 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
      121 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
      181 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      241 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      301 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      361 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      421 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      481 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      541 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      601 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      661 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      721 ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
      781 cttactggct tatcgaaatt aatacgactc actataggga gacccaagct tgcggccgct
      841 accatgggca acgccgccgc cgccaagaag ggcagcgagc aggagagcgt gaaagagttc
      901 ctagccaaag ccaaggaaga tttcctgaaa aaatgggaga ccccttctca gaatacagcc
      961 cagttggatc agtttgatag aatcaagacc cttggcaccg gctcctttgg gcgagtgatg
     1021 ctggtgaagc acaaggagag tgggaaccac tacgccatga agatcttaga caagcagaag
     1081 gtggtgaagc taaagcagat cgagcacact ctgaatgaga agcgcatcct gcaggccgtc
     1141 aacttcccgt tcctggtcaa gctggagttt tcttttaagg acaactcaaa cctgtacatg
     1201 gtcatggagt atgtagctgg tggcgagatg ttctcccacc tacggcggat tggaaggttc
     1261 agcgagcccc atgcccgttt ctacgcggcg cagatcgtcc tgacctttga gtatctgcac
     1321 tccctggacc tcatctaccg ggacctgaag cccgagaatc ttctcatcga ccagcagggc
     1381 tatattcagg tgacagactt cggttttgcc aagcgtgtga aaggccgtac ttggaccttg
     1441 tgtgggaccc ctgagtactt ggcccccgag attatcctga gcaaaggcta caacaaggct
     1501 gtggactggt gggctctcgg agtcctcatc tacgagatgg ctgctggtta cccacccttc
     1561 ttcgctgacc agcctatcca gatctatgag aaaatcgtct ctgggaaggt gcggttccca
     1621 tcccacttca gctctgactt gaaggacctg ctgcggaacc ttctgcaagt ggatctcacc
     1681 aagcgctttg ggaacctcaa gaacggggtc aatgacatca agaaccacaa gtggtttgcc
     1741 acgactgact ggattgccat ctatcagaga aaggtggaag ctcccttcat accaaagttt
     1801 aaaggccctg gggacacgag taactttgac gactatgagg aggaagagat ccgggtctcc
     1861 atcaatgaga agtgtggcaa ggagtttact gagtttatcg attatgatgt tcctgattat
     1921 gcaagcttaa tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1981 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     2041 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     2101 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     2161 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     2221 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     2281 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     2341 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     2401 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     2461 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     2521 aaccactacc tgagcaccca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac
     2581 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     2641 aagtaatcta gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc
     2701 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2761 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2821 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2881 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2941 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3001 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3061 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3121 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3181 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3241 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3301 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3361 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3421 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3481 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3541 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3601 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3661 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3721 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3781 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3841 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3901 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3961 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     4021 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4081 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4141 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4201 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4261 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4321 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     4381 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4441 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4501 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4561 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4621 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4681 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4741 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4801 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4861 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4921 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4981 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5041 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5101 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5161 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5221 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5281 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5341 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5401 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt cctcctggaa
     5461 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5521 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5581 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5641 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5701 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5761 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5821 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5881 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5941 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6001 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6061 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6121 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6181 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6241 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6301 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6361 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6421 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6481 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6541 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6601 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6661 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6721 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6781 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6841 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6901 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6961 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7021 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7081 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     7141 gtgcactctc agtacaatct gctctgatgc cg
//
