LOCUS       sequence-332636-        6281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the E12,14A mutant of the N-terminal 47 
            residues of mouse PKA catalytic subunit alpha C-terminally tagged by
            monomeric Venus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6281)
  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 6281)
  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_PKA-C-alpha-N (E12,14A)-mVenus
FEATURES             Location/Qualifiers
     source          1..6281
                     /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             1054..1080
                     /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(1081..1083,1084..1797)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1123..1144)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1384..1403)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1731..1752
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1834..1852)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1835..1852)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1872..1889)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1878..2102
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2148..2576
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2235..2254)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2445..2466
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2585..2605)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2590..2919
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2770..2905
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2832..2851
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2986..3780
                     /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(3040..3059)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3650..3669
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3954..4075
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3991..4010)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4045..4064
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4124..4140)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4124..4140)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4137..4159)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4148..4164
                     /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(4172..4178,4179..4196,4197..4202))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4217..4238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4355..4372)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4526..5114)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4606..4625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5285..6076,6077..6145))
                     /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     5908..5927
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6146..6250)
                     /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 aagggcagcg cccaggccag cgtgaaagag
      961 ttcctagcca aagccaagga agatttcctg aaaaaatggg agaccccttc tcagaataca
     1021 gcccagttgg atcagtttga tagaatcatc gattatgatg ttcctgatta tgcaagctta
     1081 atgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1141 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1201 aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1261 gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag
     1321 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1381 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1441 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1501 ctggagtaca actacaacag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc
     1561 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgccgac
     1621 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1681 ctgagctacc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1741 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagaa
     1801 ttcaaggcct ctcgagcctc tagagggccc tattctatag tgtcacctaa atgctagagc
     1861 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     1921 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     1981 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2041 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2101 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2161 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2221 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2281 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2341 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2401 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2461 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2521 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2581 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2641 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2701 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     2761 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     2821 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     2881 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     2941 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3001 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3061 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3121 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     3181 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3241 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3301 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3361 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3421 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3481 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     3541 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3601 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     3661 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     3721 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     3781 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     3841 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     3901 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     3961 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4021 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4081 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4141 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4201 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4261 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4321 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4381 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4441 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4501 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4561 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4621 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4681 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4741 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4801 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4861 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4921 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4981 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5041 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5101 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5161 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5221 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5281 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5341 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5401 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5461 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5521 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5581 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5641 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5701 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5761 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5821 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5881 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5941 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6001 gatccagttc aatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6061 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6121 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6181 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6241 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
