LOCUS       sequence-474307-        6705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a phosphorylation deficient (T to A) mutant 
            of CKAR3 under a CMV promotor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6705)
  AUTHORS   Yahiro T, Bayless-Edwards L, Jones JA, Ma L, Qin M, Mao T, Zhong H
  TITLE     A high-performance genetically encoded sensor for cellular imaging 
            of PKC activity in vivo.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 23:2024.07.19.604387. doi: 
            10.1101/2024.07.19.604387.
  PUBMED    39091834
REFERENCE   2  (bases 1 to 6705)
  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: "bioRxiv 
            [Preprint]. 2024 Jul 23:2024.07.19.604387. doi: 
            10.1101/2024.07.19.604387."
COMMENT     Sequence Label: pRK5_mutCKAR3
FEATURES             Location/Qualifiers
     source          1..6705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      994..1003
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1000..1002,1003..1005,1006..1518)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     CDS             join(2434..2436,2437..2439,2440..2952)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIK
                     VNFKIRHNIE"
     regulatory      3005..3014
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3018..3152
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3055..3074)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3109..3128
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3221..3578
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3221..3412
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3244..3264)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3429..3564
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3598..3615)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3598..3614)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3607..3629)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3827..4282
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3914..3933)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4124..4145
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4299..4318)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4418..4440
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4478..4496)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4564..4668
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4669..4737,4738..5529)
                     /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     complement(4887..4906)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5700..6288
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6189..6208
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6442..6459
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6576..6597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6612..6617,6618..6635,6636..6642)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6650..6666
                     /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)."
     primer_bind     6655..6677
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6674..6690
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6674..6690
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcgccacc atgctgcaga acgagcttgc tcttaagttg
      961 gctggacttg atattaacaa gactggaccg gtcgccacca tggtgagcaa gggcgaggag
     1021 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1081 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1141 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1201 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1261 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1321 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1381 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1441 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1501 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1561 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag
     1621 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1681 gcccgcatgc ataagttttc tcaagaacag atcggcgaaa acattgtgtg cagggtcatt
     1741 tgtaccacgg gtcaaattcc catccgagat ttgtcagctg atatttcaca agtgcttaag
     1801 gaaaaacgat ccataaagaa agtttggaca tttggtagaa acccagcctg tgactatcat
     1861 ttaggaaaca tttcaagact gtcaaataag catttccaaa tactactagg agaagacggt
     1921 aaccttttat tgaatgacat ttccactaat gggacctggt taaatgggca aaaagtcgag
     1981 aagaacagca atcagttact gtctcaaggt gatgaaataa ccgttggtgt aggcgtggaa
     2041 tcagatattt tatctctggt cattttcata aacgacaaat ttaagcagtg cctcgagcag
     2101 aacaaagttg atcgctctgc aggtaagcca ggcagcggcg agggcagcac caagggccga
     2161 ttcagaagat tccaggcgtt gaaggataag gctaaggcca ccggcggcag cgagctcatg
     2221 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2281 ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga ccccaacgag
     2341 aagcgcgatc acatggtcct gctggagaga gtgaccgccg ccgggatcac tctcggcatg
     2401 gacgagctat acaagggtgg aactggaggt tcaatggtga gcaagggcga ggagctgttc
     2461 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2521 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gctcatctgc
     2581 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg
     2641 atgtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2701 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2761 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2821 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactg gaacagcgtc
     2881 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2941 cacaacatcg agtaagcggc cgcaattccc cggggatcct ctagagtcga cctgcagaag
     3001 cttggccgcc atggcccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3061 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3121 aactcatcaa tgtatcttat catgtctgga tcgggaatta attcggcgca gcaccatggc
     3181 ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga aagaaccagc
     3241 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3301 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3361 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3421 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3481 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     3541 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctgttaaca gcttggcact
     3601 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     3661 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     3721 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     3781 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     3841 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3901 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3961 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4021 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     4081 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4141 actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg
     4201 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4261 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     4321 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4381 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4441 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     4501 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     4561 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4621 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4681 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4741 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4801 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4861 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4921 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4981 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5041 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5101 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5161 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5221 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5281 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5341 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5401 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5461 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5521 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5581 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5641 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5701 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5761 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5821 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5881 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5941 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6001 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6061 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6121 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6181 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6241 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6301 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6361 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6421 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     6481 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     6541 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     6601 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     6661 taacaatttc acacaggaaa cagctatgac atgattacga attaa
//
