LOCUS       sequence-105917-        6641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PKIalpha and nuclear localized mRuby2 (linked with IRES) 
            in a Cre-dependent manner in an AAV backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6641)
  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 6641)
  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: "Front 
            Pharmacol."; date: "2014-04-2"; volume: "5:56. doi"; pages: " 
            10.3389/fphar.2014.00056. eCollection 2014"
COMMENT     Sequence Label: AAV-FLEX-PKIalpha-IRES-nls-mRuby2
FEATURES             Location/Qualifiers
     source          1..6641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2459..2588
                     /label=AAV2 ITR
     promoter        2901..3178
                     /label=chicken beta-actin promoter
     primer_bind     3458..3480
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3542..3561
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    3596..3629
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3692..3725
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3741..4451)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             complement(4461..4481)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    4532..5083
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4693..4712)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     4922..4939
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     protein_bind    complement(5364..5397)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(5468..5501)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5543..6131
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5596..5616)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6014..6025)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6174..6295
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6185..6204)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6239..6258
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6457..6586
                     /label=AAV2 ITR
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg
     2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag
     2641 gaagatcgta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     2701 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     2761 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     2821 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     2881 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
     2941 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
     3001 gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg
     3061 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
     3121 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
     3181 agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
     3241 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
     3301 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
     3361 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg
     3421 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     3481 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     3541 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga tccccataac
     3601 ttcgtataaa gtatcctata cgaagttata tcaaaatagg aagaccaatg cttcaccatc
     3661 gacccgaatt gccaagcatc accatcgacc cataacttcg tataatgtat gctatacgaa
     3721 gttatactag gatcccctta cttgtacagc tcgtccatcc caccaccaag cccggcgaac
     3781 ttggcaactg cgtgttcgcg ttgtactacg aacatttcat tgtcactttc ctctaacctt
     3841 tccaggcggt gatcaacggc atggataccg ggcatcttga tgttcccgac ggtctttttt
     3901 gacctgtaag ttgttacgaa agagcaagac agatggccac caccatcaac tttcagtgcc
     3961 atatgagtgt atcccctcag accaccatct gctggataca tcatctctgt attaggctcc
     4021 caacccttgg tcttcttctg catcacggga ccattggagg gaaagtttac ccctctgact
     4081 tggacgtggt aaacgagaca gccatcctca aggctggtgt cctgcatgac ggtgacgact
     4141 ccaccatctt cgtatctcgt aactctttcc caagtaaaac cctcaggaaa ggactgttta
     4201 aagaaatcag gaatgccttt cgggtacttg ataaaagtac ggctgccata catgaacgac
     4261 gtggcaagaa tgtcaaaggc aaatggcagg ggtcctccct cgatgacttt gatcctcatg
     4321 gtttgagttc ccatgtacgg attgccttct ccttcacctg tgcatttgaa ttggtggccg
     4381 ttgaccgaac cttccatgac caccttcata cgcatatttt ccttgatcag ctcttcgccc
     4441 ttagacacca tggggtcttc tacctttctc ttcttttttg gaggagtaga atgttgagag
     4501 tcagcagtag cctcatcatc actagacatg gtattatcat cgtgtttttc aaaggaaaac
     4561 cacgtccccg tggttcgggg ggcctagacg ttttttaacc tcgactaaac acatgtaaag
     4621 catgtgcacc gaggccccag atcagatccc atacaatggg gtaccttctg ggcatccttc
     4681 agccccttgt tgaatacgct tgaggagagc catttgactc tttccacaac tatccaactc
     4741 acaacgtggc actggggttg tgccgccttt gcaggtgtat cttatacacg tggcttttgg
     4801 ccgcagaggc acctgtcgcc aggtgggggg ttccgctgcc tgcaaagggt cgctacagac
     4861 gttgtttgtc ttcaagaagc ttccagagga actgcttcct tcacgacatt caacagacct
     4921 tgcattcctt tggcgagagg ggaaagaccc ctaggaatgc tcgtcaagaa gacagggcca
     4981 ggtttccggg ccctcacatt gccaaaagac ggcaatatgg tggaaaataa catatagaca
     5041 aacgcacacc ggccttattc caagcggctt cggccagtaa cgttaggggg ggggggctag
     5101 cttagctttc agacttggct gcttctccct gggcttctcc actttgttcg gtggagcttc
     5161 tctgtccatc atcttcacct tctgtcttgt tgatatcaag gcctgctagt tttaaggcta
     5221 attcattgct gttgccactt gcagaggaaa ccaggatatc atgtattgca tttcttctac
     5281 ctgttcttcc tgaagcaatg aaatctgcat acgtagtttc cacatcagtc atggtggcgg
     5341 cactagtggt accgaattct agtataactt cgtataggat actttatacg aagttatcat
     5401 tgggattctt cctattttga tccaagcatc accatcgacc ctctagtcca gatctcacca
     5461 tcgacccata acttcgtata gcatacatta tacgaagtta tgtccctcga agaggttcga
     5521 attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
     5581 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     5641 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     5701 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     5761 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     5821 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     5881 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     5941 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     6001 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     6061 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     6121 cgcctccccg catcgatacc gtcgacccgg gcggccgctt cgagcagaca tgataagata
     6181 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     6241 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     6301 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag
     6361 caagtaaaac ctctacaaat gtggtaaaat cgataaggat cttcctagag catggctacg
     6421 tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg
     6481 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     6541 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc attaatgaat
     6601 cggccaacgc gcggggagag gcggtttgcg tattgggcgc t
//
