LOCUS       sequence-210727-        6975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Per2 transcription fluorescence reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6975)
  AUTHORS   Mei L, Fan Y, Lv X, Welsh DK, Zhan C, Zhang EE
  TITLE     Long-term in vivo recording of circadian rhythms in brains of freely
            moving mice.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Apr 2. pii: 1717735115. doi: 
            10.1073/pnas.1717735115.
  PUBMED    29610316
REFERENCE   2  (bases 1 to 6975)
  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: "Proc Natl 
            Acad Sci U S A. 2018 Apr 2. pii: 1717735115. doi: 
            10.1073/pnas.1717735115."
COMMENT     Sequence Label: AAV-P(Per2)-DIO-intron2-Venus-NLS-D2
FEATURES             Location/Qualifiers
     source          1..6975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   17..146
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(705..738)
                     /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(789..822)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(972..992)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(996..1706,1707..1709))
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1038..1059)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1387..1406
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1646..1667
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1719..1739)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1764..1808)
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             complement(1830..1895)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(2622..2645)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2645..2654
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2692..2710)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2693..2710)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(2716..2734)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2777..2796)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          2779..2911
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     protein_bind    2976..3009
                     /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    3060..3093
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3118..3706
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3171..3191)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3589..3600)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3738..4214
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3937..3956)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4254..4394
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4254..4383
                     /label=AAV2 ITR
     rep_origin      4469..4924
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4556..4575)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4766..4787
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4941..4960)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5060..5082
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5120..5138)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5206..5310
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5311..5379,5380..6171)
                     /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(5529..5548)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6342..6930
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6831..6850
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg
       61 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt
      121 ggccaactcc atcactaggg gttcctgcgg ccgcacgcgt cggattaccg aggctggtca
      181 cgtcgtcgca ggtgataggc cgggggccct ggtctctgcc ggctgtgagt tgcgcagcgg
      241 ccaagcacca ttcccccgcg ccgcagtggt acgcgccact ccggggctgc acgagcgggc
      301 caccgccgtg ccaggtgaat ggaagtcccg caggccggaa gtggacgagc ctactcgccc
      361 gggcgcgggg gggcgcaaga gcgcgcagca tcttcattga ggaacccggg cggcgaacat
      421 ggagttccat gtgcgtctta tgtaaagaga gcgacgggcg tctccaccaa ttgacgagcg
      481 tagctctcag gttccgcccc gccagtatgc aaatgaggtg gcactccgac caatggcgcg
      541 cgcaggggcg ggctcagcgc gcgcggtcac gttttccact atgtgacagc ggagggcgac
      601 gcggcggcag cggcgctact gggactagcg gctccgggcg gctgcggcgc aggccgagcg
      661 caccaagtga cgggccgagc aagggatcta gagtcgactc cggaataact tcgtatagga
      721 tactttatac gaagttatgc agaatggtag ctggattgta gctgctatta gcaatatgaa
      781 acctcttaat aacttcgtat agcatacatt atacgaagtt atggctacac attgatccta
      841 gcagaagcac aggctgcagg gtgacggtcc atcccgctct cctgggcaca agacatgggc
      901 agcgtgccat catcctgctc ctccacctcc ggcgggaagc catggctaag cttagaattc
      961 acggggtctt ctacctttct cttctttttt ggaggcttgt acagctcgtc catgccgaga
     1021 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg
     1081 tctttgctca gtttggactg gtagctcagg tagtggttgt cgggcagcag cacggggccg
     1141 tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgccgcc gtcctcgatg
     1201 ttgtggcgga tcttgaagtt tgccttgatg ccgttcttct gcttgtcggc ggtgatatag
     1261 acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg
     1321 aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg
     1381 gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct
     1441 tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcgggcgaag
     1501 cactgcaggc cgtagccgag ggtggtcacg agggtgggcc agggcacggg cagcttgccg
     1561 gtggtgcaga tcaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg
     1621 gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc
     1681 ccggtgaaca gctcctcgcc cttgctcaca cctgcagggc cctgaaaata caggttttcg
     1741 gatcctgaac cgcctgccga tccctcatgc cactctattt tctgagcttc gaaaatatcg
     1801 ttcaggccgc tagctgcggc cgctccaccc ttatcgtcat cgtccttgta gtcgatgtca
     1861 tgatctttat aatcaccgtc atggtctttg tagtcctgca aaggaagata gatagataag
     1921 atctccacac gcctcttccc ccaaccagcc gataaaactg accagggaca tcacaccaga
     1981 gattttaaag aaaatattac cgtatgctgg gtggtggctt tgggacaaag gacttgcctg
     2041 gagtgcctga gacccatagt atggcctcca tacttcccct caaaataaaa gtcagagctg
     2101 gagaatcagc tcaatggtga agagaagcac ccacacggct gcttacaccc atctgtaact
     2161 cacagttcca ggggatccaa tgccctcttc tgtcccctat ggtcaccaaa catgcaagtg
     2221 gtgcaccgac atacatacag gcaaaacacc catatacttt aaagtaaaaa tttaaaaaag
     2281 gaaaagtcat gttaaaactg aaacataaga gcttcagcta ttaggagact tgaacactga
     2341 gagacagaaa tgaaaaggaa tattctgtga tcgaatgcat gtgtgtgagc aggctcctcc
     2401 gaggtcttac tggactgtct gttaagtgca tcagatacaa aaatgcaaag atgacgtggt
     2461 atcgttattt ggaaggacaa agtacaataa ataccatcta caggattaag aagtactgcc
     2521 atcctgcaag tcctggtact tgctgcttga agacgggcga caaactatgg agggagccac
     2581 ccgtgtcctg tgtgcgaggg cctggcctga ggctgaccta ccttgtcatc gtcatccttg
     2641 tagtccatgg tggcctctcc ttcgagtgca aaaagaacaa gtagcttgta ttctatagtg
     2701 tcacctaaat gctagcctat agtgagtcgt attaagtact ctagccttaa gagctgtaat
     2761 tgaactggga gtggacacct gtggagagaa aggcaaagtg gatgtcagta agaccaatag
     2821 gtgcctatca gaaacgcaag agtcttctct gtctcgacaa gcccagtttc tattggtctc
     2881 cttaaacctg tcttgtaacc ttgatactta cctgcccagt gcctcacgac caactttcgc
     2941 taccttagga ccgttatagt tactgcagcg ctagcataac ttcgtataaa gtatcctata
     3001 cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa
     3061 taacttcgta taatgtatgc tatacgaagt tatgaattcg atatcaagct tatcgataat
     3121 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     3181 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3241 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3301 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3361 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3421 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3481 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3541 tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3601 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3661 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg
     3721 ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc
     3781 tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt
     3841 tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg
     3901 ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg
     3961 cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc
     4021 ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt
     4081 ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc
     4141 aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc
     4201 ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc
     4261 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4321 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     4381 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4441 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     4501 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     4561 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4621 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4681 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4741 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4801 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     4861 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     4921 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     4981 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5041 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5101 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     5161 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5221 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5281 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5341 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5401 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5461 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5521 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5581 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5641 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5701 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5761 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5821 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5881 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5941 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6001 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6061 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6121 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6181 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6241 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6301 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6361 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6421 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6481 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6541 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6601 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6661 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6721 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6781 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6841 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6901 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6961 ctggcctttt gctgg
//
