LOCUS       sequence-224226-        7343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of rat PKA regulatory subunit II-beta 
            C-terminally tagged by sREACh..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7343)
  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 7343)
  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-RII-beta-sREACh
FEATURES             Location/Qualifiers
     source          1..7343
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        165..544
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        545..748
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     699..719
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     793..812
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        793..811
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2091..2093,2094..2096,2097..2807)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     ERVTAAGITLGMDELYK"
     primer_bind     complement(2136..2157)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2397..2416)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(2826..2844)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2827..2844)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2864..2881)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2870..3094
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3140..3568
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3227..3246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3437..3458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3577..3597)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3582..3911
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3762..3897
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3824..3843
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3978..4772
                     /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(4032..4051)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4642..4661
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4946..5067
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4983..5002)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5037..5056
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5116..5132)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5116..5132)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5129..5151)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5140..5156
                     /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(5164..5170,5171..5188,5189..5194))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5209..5230
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5347..5364)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5518..6106)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5598..5617)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6277..7068,7069..7137))
                     /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     6900..6919
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7138..7242)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7317..7336)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
       61 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
      121 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      181 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      241 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      301 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      361 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      421 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      481 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      541 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      601 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      661 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      721 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      781 gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc gccaccatga
      841 gcatcgagat ccccgcgggg ctcacggagc tgctgcaggg cttcacggtg gaggtgctga
      901 ggcaccagcc cgccgacctg ctggagttcg cgctgcagca cttcacgcgg ctgcagcagg
      961 agaacgagcg caagggcgcc gcgcgcttcg gccatgaggg caggacctgg ggggacgcgg
     1021 gcgcagccgc cgggggcgga acccccagta agggtgtcaa cttcgcggag gagcccatgc
     1081 gctccgattc cgagaacggc gaagaggagg aggccgcgga agcaggggcg ttcaacgctc
     1141 cagttataaa ccggttcaca aggcgtgcct cggtatgtgc agaagcttat aatcctgatg
     1201 aagaagaaga tgatgcagag tccaggataa tacatcccaa aactgacgat caaagaaaca
     1261 gattgcaaga agcctgcaaa gacatcctgc tgtttaagaa cctggatcca gaacagatgt
     1321 ctcaagtatt agatgccatg tttgaaaaat tggtcaaaga aggggaacac gtaatcgatc
     1381 aaggtgatga tggtgacaac ttttacgtca tcgacagagg aacatttgat atttatgtaa
     1441 aatgtgatgg cgttggaaga tgcgttggta actatgacaa tcgtgggagt tttggagaac
     1501 tggccttaat gtacaataca cccagagcag ctacaatcac tgctacctct cctggtgctc
     1561 tgtggggttt ggacagggtg accttcagga gaataatagt aaaaaacaat gccaaaaaga
     1621 ggaagatgta cgagagcttt attgagtcac tgccattcct caagtctctg gaggtttctg
     1681 aacgcctgaa ggtggtagat gtgattggca ccaaagttta caacgatgga gaacagatca
     1741 ttgctcaggg agactcggcg gattcgttct tcattgtaga atctggagaa gtgagaatta
     1801 ctatgaagag aaagggtaaa tcagacatcg aagagaacgg tgctgtggaa atcgctcggt
     1861 gtctccgggg acagtatttt ggagagcttg ccctggtcac taacaagcca agagcagcat
     1921 ctgcacacgc cattgggact gtcaaatgct tagccatgga tgtgcaagca tttgagaggc
     1981 ttctgggacc ttgcatggaa attatgaaga ggaatatcgc cacctatgag gagcaattag
     2041 ttgcactgtt tgggacaaac atggatattg ttgagcccac tgcagatatc atggtgagca
     2101 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2161 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2221 ccctgaagct catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2281 ccttcggcta cggcctgatg tgcttcgccc gctaccccga ccacatgaag cagcacgact
     2341 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2401 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2461 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2521 acaactggaa cagcgtcaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2581 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2641 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct
     2701 accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggaga
     2761 gagtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa tctagagggc
     2821 cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct
     2881 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2941 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3001 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3061 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3121 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3181 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3241 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3301 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3361 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3421 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3481 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3541 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     3601 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3661 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3721 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3781 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3841 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3901 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     3961 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     4021 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4081 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4141 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4201 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4261 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4321 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4381 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4441 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4501 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4561 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4621 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4681 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4741 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4801 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4861 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4921 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4981 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5041 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     5101 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5161 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5221 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5281 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5341 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5401 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5461 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5521 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5581 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5641 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5701 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5761 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5821 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5881 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcccaa
     5941 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6001 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6061 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6121 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6181 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6241 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6301 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6361 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6421 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6481 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6541 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6601 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6661 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6721 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6781 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6841 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6901 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6961 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7021 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7081 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7141 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7201 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7261 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
     7321 atctgctctg atgccgcata gtt
//
