LOCUS       sequence-399618-        6840 bp    DNA     circular SYN 15-APR-2025
DEFINITION  An adeno-associated viral vector expressing Cre-dependent 
            channelrhodopsin-2 fused to a V5 epitope and the ligand-binding 
            domain of estrogen receptor (ChRERa) from a synapsin promoter, for 
            use in PET.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6840)
  AUTHORS   Bonaventura J, Boehm MA, Jedema HP, Solis O, Pignatelli M, Song X, 
            Lu H, Richie CT, Zhang S, Gomez JL, Lam S, Morales M, Gharbawie OA, 
            Pomper MG, Stein EA, Bradberry CW, Michaelides M
  TITLE     Expression of the excitatory opsin ChRERalpha can be traced 
            longitudinally in rat and nonhuman primate brains with PET imaging.
  JOURNAL   Sci Transl Med. 2023 Jul 26;15(706):eadd1014. doi: 
            10.1126/scitranslmed.add1014. Epub 2023 Jul 26.
  PUBMED    37494470
REFERENCE   2  (bases 1 to 6840)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Transl 
            Med."; date: "2023-07-26"; volume: "15(706):eadd1014. doi"; pages: "
            10.1126/scitranslmed.add1014. Epub 2023 Jul 26"
COMMENT     Sequence Label: pOTTC1822 - pAAV SYN1 DIO hChR2(H134R)-V5-ER-L...
FEATURES             Location/Qualifiers
     source          1..6840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /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(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(793..1686)
                     /codon_start=1
                     /gene="ESR1"
                     /product="estrogen nuclear receptor alpha ligand-binding 
                     domain"
                     /label=ER-LBD
                     /translation="KRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM
                     GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKL
                     LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF
                     LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNK
                     GMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSL
                     QKYYITGEAEGFPATV"
     CDS             complement(799..1734)
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             complement(1780..1821)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1888..2814)
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     protein_bind    2825..2858
                     /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    2909..2942
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2967..3555
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3020..3040)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3438..3449)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3587..4063
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3786..3805)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4103..4243
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4103..4232
                     /label=AAV2 ITR
     rep_origin      4318..4773
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4405..4424)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4615..4636
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4790..4809)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4909..4931
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4969..4987)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5055..5159
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5160..5228,5229..6020)
                     /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(5378..5397)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6191..6779
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6680..6699
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgcccacct gcctagactg tggcagggaa accctctgcc tcccccgtga tgtaatactt
      841 ttgcaaggaa tgcgatgaag tagagcccgc agtggccaag tggctttggt ccgtctcctc
      901 cacggatgcc cctccacggc tagtgggcgc atgtaggcgg tgggcgtcca gcatctccag
      961 cagcaggtca tagaggggca ccacgttctt gcacttcatg ctgtacagat gctccatgcc
     1021 tttgttactc atgtgcctga tgtgggagag gatgaggagg agctgggcca gccgctggtg
     1081 ctgctgctgc agggtcaggc ctgccttggc catcaggtgg atcaaagtgt ctgtgatctt
     1141 gtccaggact cggtggatat ggtccttctc ttccagagac ttcagggtgc tggacagaaa
     1201 tgtgtacact ccagaattaa gcaaaataat agatttgagg cacacaaact cctctccctg
     1261 cagattcatc atgcggaacc gagatgatgt agccagcagc atgtcgaaga tctccaccat
     1321 gccctctaca cattttccct ggttcctgtc caagagcaag ttaggagcaa acagtagctt
     1381 ccctgggtgc tccatggagc gccagacgag gccaatcatc aggatctcta gccaggcaca
     1441 ttccagaagg tggacctgat catggagggt caaatccaca aagcctggca ccctcttcgc
     1501 ccagttgatc atgtgaacca gctccctgtc tgccaggttg gtcagtaagc ccatcatgga
     1561 ggcttcactg aagggtctgg taggatcata ctcggaatag agtatggggg gctcagcatc
     1621 caacaaggca ctgaccatct ggtcggccgt cagggacaag gccaggctgt tcttcttaga
     1681 gcgtttgatc atgagcgggc ttggccaaag gttggcagct ctcatgtctc cagcagatga
     1741 acctgaacca gtggcccatg gacaaccgcc gccggttcta gttgagtcca agcccagcag
     1801 aggattgggt attggctttc cattcacggc atcgaacccc ttaatatgac agtaagcttc
     1861 catagatcca ccggtggcgg ccgctggcac ggctccggcc tcggcttcgt cttcgacgag
     1921 agtctcgacc tcgatctccg ttccgccgat gttcagtttg gtggttttgc ggatatctcc
     1981 gtgaatcaat atgtgctcgt ggatcaggac gcgcaggtag tgtcccaaca acccccaaca
     2041 atttttactc atcagatcaa taatcgtgtg acctacggtg gagccataga cgctcaggac
     2101 gccaaaacct tcgggcccca aaatgaagag aattgggaac ataccccagc tcacgaaaaa
     2161 cagccatgcc atgccggtca cgacctggcg gcaccgaccc tttggcacag tatgataacc
     2221 ctcgatatat gctttggcgg cgtgaaaaaa tgtgttcgcg ccatagcaca atccaagaca
     2281 aaagaagatg actttaacat agccggttgc catggcgctg gtagcccccc acacgatagt
     2341 cccgatgtct gagacaagga gtcccatggt tctcctgctg tagtcgttgc tcaggccggt
     2401 gaggttgctc aggcggataa ggatgacagg acaagtgagc agccactctg catagcgcag
     2461 ccactgcacc cggtgtcctg tggcaaggta gagcatagag ggattcttaa actcaaaaaa
     2521 gaactcgaga atcaccttaa ccatttcaat ggcgcacaca tagatctcct cccagccgca
     2581 tgtagatttc caggtttggt aggcatagaa catcagcagc aaaatgctga atcctgctgc
     2641 aagccactgc aggacatttg acgcggtctg agcgccgttc gtgccgcgag attcaatcca
     2701 tccggcacag taacattgat cctcagggac caggacggac ccgttcacca ccacaggatt
     2761 agtaacgaac aaaagttcgc gtccgacggc agacaaagcg ccgccatagt ccatggtgct
     2821 agcgataact tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc
     2881 actgttattc tttagaatgg tgcaaagaat aacttcgtat aatgtatgct atacgaagtt
     2941 atgaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     3001 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3061 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     3121 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     3181 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     3241 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3301 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     3361 aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt
     3421 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     3481 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     3541 gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt ggcatccctg
     3601 tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct
     3661 tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat
     3721 ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg
     3781 cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc
     3841 cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc
     3901 atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt
     3961 ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt
     4021 gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac
     4081 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     4141 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4201 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     4261 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     4321 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4381 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4441 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4501 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4561 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4621 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     4681 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4741 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     4801 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4861 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4921 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     4981 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     5041 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5101 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5161 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5221 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5281 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5341 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5401 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5461 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5521 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5581 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5641 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5701 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5761 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5821 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5881 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5941 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6001 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6061 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6121 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6181 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6241 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6301 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6361 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6421 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6481 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6541 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6601 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6661 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6721 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6781 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
