LOCUS       sequence-332602-        7172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the E12,14K mutant of the mouse PKA 
            catalytic subunit alpha C-terminally tagged by monomeric EGFP. 
            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   Xiong WH, Qin M, Zhong H
  TITLE     Myristoylation alone is sufficient for PKA catalytic subunits to 
            associate with the plasma membrane to regulate neuronal functions.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Apr 13;118(15). pii: 2021658118. doi:
            10.1073/pnas.2021658118.
  PUBMED    33876760
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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2021-04-13"; volume: "118(15). pii: 
            2021658118. doi"; pages: " 10.1073/pnas.2021658118"
COMMENT     Sequence Label: pCDNA3_shRNA resistant PKA-C-alpha(E12,14K)-mEGFP
FEATURES             Location/Qualifiers
     source          1..7172
                     /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             1966..1992
                     /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(1993..1995,1996..2709)
                     /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(2035..2056)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2296..2315)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2643..2664
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2725..2743)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2726..2743)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2763..2780)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2769..2993
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3039..3467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3126..3145)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3336..3357
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3476..3496)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3481..3810
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3661..3796
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3723..3742
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3877..4671
                     /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(3931..3950)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4541..4560
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4845..4966
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4882..4901)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4936..4955
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5015..5031)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5015..5031)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5028..5050)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5039..5055
                     /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(5063..5069,5070..5087,5088..5093))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5108..5129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5246..5263)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5417..6005)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6176..6967,6968..7036))
                     /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     6799..6818
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7037..7141)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 gcttgcggcc
      901 gctaccatgg gcaacgccgc cgccgccaag aagggcagca agcagaagag cgtgaaagag
      961 ttcctagcca aagccaagga agatttcctg aaaaaatggg agaccccttc tcagaataca
     1021 gcccagttgg atcagtttga tagaatcaag acccttggca ccggctcctt tgggcgagtg
     1081 atgctggtga agcacaagga gagtgggaac cactacgcca tgaagatctt agacaagcag
     1141 aaggtggtga agctaaagca gatcgagcac actctgaatg agaagcgcat cctgcaggcc
     1201 gtcaacttcc cgttcctggt caagctggag ttttctttta aggacaactc aaacctgtac
     1261 atggtcatgg agtatgtagc tggtggcgag atgttctccc acctacggcg gattggaagg
     1321 ttcagcgagc cccatgcccg tttctacgcg gcgcagatcg tcctgacctt tgagtatctg
     1381 cactccctgg acctcatcta ccgggacctg aagcccgaga atcttctcat cgaccagcag
     1441 ggctatattc aggtgacaga cttcggtttt gccaagcgtg tgaaaggccg tacttggacc
     1501 ttgtgtggga cccctgagta cttggccccc gagattatcc tgagcaaagg ctacaacaag
     1561 gctgtggact ggtgggctct cggagtcctc atctacgaga tggctgctgg ttacccaccc
     1621 ttcttcgctg accagcctat ccagatctat gagaaaatcg tctctgggaa ggtgcggttc
     1681 ccatcccact tcagctctga cttgaaggac ctgctgcgga accttctgca agtggatctc
     1741 accaagcgct ttgggaacct caagaacggg gtcaatgaca tcaagaacca caagtggttt
     1801 gccacgactg actggattgc catctatcag agaaaggtgg aagctccctt cataccaaag
     1861 tttaaaggcc ctggggacac gagtaacttt gacgactatg aggaggaaga gatccgggtc
     1921 tccatcaatg agaagtgtgg caaggagttt actgagttta tcgattatga tgttcctgat
     1981 tatgcaagct taatgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2041 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2101 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     2161 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     2221 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2281 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2341 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2401 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2461 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2521 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2581 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat
     2641 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2701 tacaagtaat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     2761 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2821 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2881 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2941 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3001 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3061 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3121 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3181 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3241 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3301 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3361 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3421 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3481 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3541 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3601 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3661 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3721 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3781 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3841 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3901 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3961 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4021 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4081 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4141 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4201 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4261 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4321 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4381 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4441 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4501 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4561 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4621 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4681 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4741 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4801 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4861 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4921 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4981 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5041 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5101 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5161 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5221 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5281 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5341 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5401 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5461 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttcctcctg
     5521 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5581 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5641 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5701 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5761 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5821 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5881 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5941 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6001 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6061 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6121 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6181 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6241 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6301 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6361 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6421 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6481 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6541 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6601 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6661 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6721 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6781 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6841 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6901 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6961 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7021 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7081 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7141 gcacatttcc ccgaaaagtg ccacctgacg tc
//
