LOCUS       sequence-235218-        7269 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of the tAKAR4-alpha sensor for detecting PKA 
            activity using ratiometric imaging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7269)
  AUTHORS   Ma L, Jongbloets BC, Xiong WH, Melander JB, Qin M, Lameyer TJ, 
            Harrison MF, Zemelman BV, Mao T, Zhong H
  TITLE     A Highly Sensitive A-Kinase Activity Reporter for Imaging 
            Neuromodulatory Events in Awake Mice.
  JOURNAL   Neuron. 2018 Aug 22;99(4):665-679.e5. doi: 
            10.1016/j.neuron.2018.07.020. Epub 2018 Aug 9.
  PUBMED    30100256
REFERENCE   2  (bases 1 to 7269)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.neuron.2018.07.020"; journalName: "Neuron"; date: 
            "2018-08-22- 22"; volume: "99"; issue: "4"; pages: "665-679.e5"
COMMENT     Sequence Label: pRK5_tAKAR4-alpha
FEATURES             Location/Qualifiers
     source          1..7269
                     /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      943..952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(949..951,952..954,955..1665)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2188..2922
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     regulatory      3569..3578
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3582..3716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3619..3638)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3673..3692
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3785..4142
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3785..3976
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3808..3828)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3993..4128
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4162..4179)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4162..4178)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4171..4193)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4391..4846
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4478..4497)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4688..4709
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4863..4882)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4982..5004
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5042..5060)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5128..5232
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5233..5301,5302..6093)
                     /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(5451..5470)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6264..6852
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6753..6772
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7006..7023
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7140..7161
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7176..7181,7182..7199,7200..7206)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7214..7230
                     /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     7219..7241
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7238..7254
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7238..7254
                     /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 attcccgggc tcgagaccgg tcgccaccat ggtgagcaag
      961 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1021 ggccacaggt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1081 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1141 ctgacctggg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1201 ttcaagtccg ccatgcccga aggctacgtc caggagcgta ccatcttctt caaggacgac
     1261 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1321 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1381 aacgccatca gtgacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1441 cacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1501 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1561 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1621 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagcgcat gcataagttt
     1681 tctcaagaac agatcggcga aaacattgtg tgcagggtca tttgtaccac gggtcaaatt
     1741 cccatccgag atttgtcagc tgatatttca caagtgctta aggaaaaacg atccataaag
     1801 aaagtttgga catttggtag aaacccagcc tgtgactatc atttaggaaa catttcaaga
     1861 ctgtcaaata agcatttcca aatactacta ggagaagacg gtaacctttt attgaatgac
     1921 atttccacta atgggacctg gttaaatggg caaaaagtcg agaagaacag caatcagtta
     1981 ctgtctcaag gtgatgaaat aaccgttggt gtaggcgtgg aatcagatat tttatctctg
     2041 gtcattttca taaacgacaa atttaagcag tgcctcgagc agaacaaagt tgatcgctct
     2101 gcaggtaagc caggcagcgg cgagggcagc accaagggcc tgcgtcgcgc caccctggtg
     2161 gacggcggca ccggcggcag cgagctcatg ggcggcgtgc agctcgccga ccactaccag
     2221 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     2281 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2341 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggagg taccggtgga
     2401 tctatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2461 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2521 tacggcaagc tgaccctgaa gctgatctgc accaccggca agctgcccgt gccctggccc
     2581 accctcgtga ccaccctggg ctacggcctt cagtgcttcg cccgctaccc cgaccacatg
     2641 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2701 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2761 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2821 cacaagctgg agtacaacta caacagccac aacgtctata tcaccgccga caagcagaag
     2881 aacggcatca aggccaactt caagatccgc cacaacatcg agtccggagt gtacaagggt
     2941 accccaggaa cccccaagtc tgccatcttg gtgccgagtg agaagaaggt cgccatcata
     3001 cgtactcctc caaaatctcc tgcgactccc aagcagcttc ggcttattaa ccaaccactg
     3061 ccagacctga agaatgtcaa atccaaaatc ggatcaacag acaacatcaa ataccagcct
     3121 aaaggggggc aggtacaaat tgttaccaag aaaatagacc taagccatgt gacatccaaa
     3181 tgtggctctc tgaagaacat ccgccacagg ccaggtggcg gacgtgtgaa aattgagagt
     3241 gtaaaactag atttcaaaga aaaggcccaa gctaaagttg gttctcttga taatgctcat
     3301 catgtacctg gaggtggtaa tgtcaagatt gacagccaaa agttgaactt cagagagcat
     3361 gctaaagccc gtgtggacca tggggctgag atcattacac agtccccagg cagatccagc
     3421 gtggcatcac cccgacgact cagcaatgtc tcctcgtctg gaagcatcaa cctgctcgaa
     3481 tctcctcagc ttgccacttt ggctgaggat gtcactgctg cactcgctaa gcagggcttg
     3541 taatctagag tcgacctgca gaagcttggc cgccatggcc caacttgttt attgcagctt
     3601 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3661 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcggga
     3721 attaattcgg cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta
     3781 ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3841 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3901 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3961 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4021 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4081 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4141 aaaagctgtt aacagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     4201 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     4261 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     4321 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4381 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4441 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4501 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4561 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4621 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4681 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct
     4741 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4801 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     4861 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4921 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4981 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5041 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5101 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5161 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5221 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5281 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5341 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5401 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5461 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5521 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5581 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5641 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5701 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5761 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5821 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5881 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5941 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6001 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6061 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6121 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6181 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6241 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6301 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6361 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6421 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6481 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6541 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6601 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6661 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6721 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6781 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6841 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6901 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6961 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     7021 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     7081 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     7141 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     7201 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt
     7261 acgaattaa
//
