LOCUS       sequence-105903-        7279 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses the Fluorescence Lifetime Imaging Microscopy 
            (FLIM)-compatible PKA activity sensor FLIM-AKAR in an AAV backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7279)
  AUTHORS   Chen Y, Saulnier JL, Yellen G, Sabatini BL
  TITLE     A PKA activity sensor for quantitative analysis of endogenous GPCR 
            signaling via 2-photon FRET-FLIM imaging.
  JOURNAL   Front Pharmacol. 2014 Apr 2;5:56. doi: 10.3389/fphar.2014.00056. 
            eCollection 2014.
  PUBMED    24765076
REFERENCE   2  (bases 1 to 7279)
  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"; journalName: "Front 
            Pharmacol."; date: "2014-04-2"; volume: "5:56. doi"; pages: " 
            10.3389/fphar.2014.00056. eCollection 2014"
COMMENT     Sequence Label: AAV-FLIM-AKAR
FEATURES             Location/Qualifiers
     source          1..7279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1432..1441
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1438..1440,1441..1443,1444..1956)
                     /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"
     primer_bind     complement(1483..1504)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    3406..3994
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3459..3479)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3877..3888)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4026..4502
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4225..4244)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4542..4682
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4542..4671
                     /label=AAV2 ITR
     rep_origin      4757..5212
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4844..4863)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5054..5075
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5229..5248)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5348..5370
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5408..5426)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5494..5598
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5599..5667,5668..6459)
                     /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(5817..5836)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6630..7218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7119..7138
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc cgccaccatg
     1441 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1501 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1561 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1621 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1681 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1741 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1801 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1861 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1921 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctggctgat
     1981 cattaccagc agaacacacc tatcggagac gggcccgtcc tgctccctga taatcattat
     2041 ctgtctactc agagtaagct ctcaaaagac ccaaacgaga aacgggatca catggtgctg
     2101 ctcgagttcg tcactgccgc tcgcatgcat aagttttccc aggagcagat cggtgaaaat
     2161 atcgtgtgcc gggtcatctg tacaactgga cagatcccca ttcgcgacct gtctgccgac
     2221 atcagtcagg tgctgaagga gaaaaggtct attaagaaag tctggacatt cggaagaaac
     2281 cccgcttgcg actatcacct ggggaacatt agcaggctgt ccaataagca ctttcagatt
     2341 ctcctgggcg aagacgggaa cctcctcctc aacgacatca gcactaacgg gacctggtta
     2401 aatgggcaaa aagtcgagaa gaacagcaat cagttactgt ctcaaggtga tgaaataacc
     2461 gttggtgtag gcgtggaatc agatatttta tctctggtca ttttcataaa cgacaaattt
     2521 aagcagtgcc tcgagcagaa caaagttgat cgctctgcag gtaagccagg cagcggcgag
     2581 ggcagcacca agggcctgcg tcgcgccacc ctggtggacg gcggcaccgg cggctcagaa
     2641 ctcatgggtt cagtccagct cgcagaccac tatcagcaga atactccaat cggtgacggg
     2701 ccagtcctcc tcccagacaa ccactacctc tcttatcaga gtgccctgtc aaaagatcca
     2761 aatgagaaaa gggatcatat ggtgctgctc gaaagagtca cagccgctgg aatcactctc
     2821 gggatggacg agctgtacaa aggcggaaca gggggtagca tggtgtccaa gggcgaggaa
     2881 ctgttcactg gcgtggtccc cattctcgtg gaactggacg gcgacgtgaa cggccacaag
     2941 ttcagcgtga gcggcgaggg agaaggggat gctacctatg gaaagctgac actcaaactg
     3001 atctgcacca cagggaagct gcccgtgcct tggccaaccc tggtcactac cttcggttac
     3061 ggcctgatgt gctttgcaag gtaccccgac cacatgaagc agcatgattt ctttaaatct
     3121 gccatgcctg agggatacgt gcaggaacgg acaattttct ttaaggacga tggcaactac
     3181 aaaactcgcg cagaggtgaa gttcgaaggt gacaccctcg tcaaccgaat cgagctgaag
     3241 gggattgact ttaaagaaga tggaaatatc ctcgggcata agctggagta caactggaat
     3301 agcgtgaacg tctatattat ggccgacaag cagaaaaacg gtatcaaggt caacttcaaa
     3361 atcaggcaca acatcgagta agaattcgat atcaagctta tcgataatca acctctggat
     3421 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3481 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3541 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     3601 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3661 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3721 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3781 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc
     3841 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3901 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3961 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga
     4021 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
     4081 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg
     4141 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga
     4201 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat
     4261 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg
     4321 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
     4381 gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc
     4441 caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc
     4501 ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     4561 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     4621 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     4681 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4741 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4801 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4861 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4921 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4981 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5041 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc
     5101 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5161 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     5221 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5281 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5341 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5401 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     5461 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5521 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5581 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5641 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5701 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5761 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5821 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5881 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5941 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6001 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6061 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6121 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6181 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6241 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6301 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6361 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6421 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6481 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6541 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6601 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6661 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6721 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     6781 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6841 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6901 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6961 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7021 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7081 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7141 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7201 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7261 tggccttttg ctcacatgt
//
