LOCUS       sequence-329412-        6292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cre-inactivated ChR2-TdTomato under control of the TRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6292)
  AUTHORS   Sabatini PV, Wang J, Rupp AC, Affinati AH, Flak JN, Li C, Olson DP, 
            Myers MG Jr
  TITLE     tTARGIT AAVs mediate the sensitive and flexible manipulation of 
            intersectional neuronal populations in mice.
  JOURNAL   eLife. 2021 Mar 11;10. pii: 66835. doi: 10.7554/eLife.66835.
  PUBMED    33704065
REFERENCE   2  (bases 1 to 6292)
  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: "eLife."; 
            date: "2021-03-11"; volume: "10. pii: 66835. doi"; pages: " 
            10.7554/eLife.66835"
COMMENT     Sequence Label: pAAV-TRE-DIO-ChR2-TdTomato (Cre-OFF)
FEATURES             Location/Qualifiers
     source          1..6292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   531..671
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   542..671
                     /label=AAV2 ITR
     protein_bind    join(828..846,847..869,870..888,889..911,912..930,931..953,
                     954..972,973..995,996..1014,1015..1037,1038..1056,
                     1057..1079,1080..1098)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        1131..1169
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    complement(1304..1337)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1348..1381)
                     /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)."
     CDS             1448..2374
                     /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"
     regulatory      2384..2393
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2390..3100
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2535..2553)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2803..2822
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2997..3020
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    3136..3169
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3180..3213
                     /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)."
     misc_feature    3237..3825
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3290..3310)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3708..3719)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3860..3877)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3866..4073
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4096..4225
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4287..4875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4367..4386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5046..5837,5838..5906))
                     /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     5669..5688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5907..6011)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6079..6097
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6135..6157)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6257..6276
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca atagaccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga
      121 tagagttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg
      481 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc cctgcaggca
      541 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
      601 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      661 taggggttcc tgcggccgca cgcgaaacaa ttctgcagga attcggggcc gcggaggctg
      721 gatcggtccc ggtgtcttct atggaggtca aaacagcgtg gatggcgtct ccaggcgatc
      781 tgacggttca ctaaacgagc tctgcttata taggtcgagt ttaccactcc ctatcagtga
      841 tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc
      901 gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat
      961 cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg
     1021 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact
     1081 ccctatcagt gatagagaaa agtgaaagtc gagctcggta cccgggtcga ggtaggcgtg
     1141 tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac
     1201 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggcc
     1261 ccgaattcga gctcggtacc cggggatcct ctagtcgacg tcgataactt cgtatagcat
     1321 acattatacg aagttatccg aatcgcaata acttcgtata ggatacttta tacgaagtta
     1381 tttggcgcgc caaatttaaa taacgcaacg taccttcgag ggggcccaag gaattcttac
     1441 ttgtacgatg gactatggcg gcgctttgtc tgccgtcgga cgcgaacttt tgttcgttac
     1501 taatcctgtg gtggtgaacg ggtccgtcct ggtccctgag gatcaatgtt actgtgccgg
     1561 atggattgaa tctcgcggca cgaacggcgc tcagaccgcg tcaaatgtcc tgcagtggct
     1621 tgcagcagga ttcagcattt tgctgctgat gttctatgcc taccaaacct ggaaatctac
     1681 atgcggctgg gaggagatct atgtgtgcgc cattgaaatg gttaaggtga ttctcgagtt
     1741 cttttttgag tttaagaatc cctctatgct ctaccttgcc acaggacacc gggtgcagtg
     1801 gctgcgctat gcagagtggc tgctcacttg tcctgtcatc cttatccgcc tgagcaacct
     1861 caccggcctg agcaacgact acagcaggag aaccatggga ctccttgtct cagacatcgg
     1921 gactatcgtg tggggggcta ccagcgccat ggcaaccggc tatgttaaag tcatcttctt
     1981 ttgtcttgga ttgtgctatg gcgcgaacac attttttcac gccgccaaag catatatcga
     2041 gggttatcat actgtgccaa agggtcggtg ccgccaggtc gtgaccggca tggcatggct
     2101 gtttttcgtg agctggggta tgttcccaat tctcttcatt ttggggcccg aaggttttgg
     2161 cgtcctgagc gtctatggct ccaccgtagg tcacacgatt attgatctga tgagtaaaaa
     2221 ttgttggggg ttgttgggac actacctgcg cgtcctgatc cacgagcaca tattgattca
     2281 cggagatatc cgcaaaacca ccaaactgaa catcggcgga acggagatcg aggtcgagac
     2341 tctcgtcgaa gacgaagccg aggccggagc cgtggcaccg gtcgccacca tggtgagcaa
     2401 gggcgaggag gacaacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
     2461 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     2521 gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga
     2581 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     2641 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     2701 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     2761 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     2821 gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     2881 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgccg aggtcaagac
     2941 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagct
     3001 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc gcgccgaggg
     3061 ccgccactcc accggcggca tggacgagct gtacaagtaa gctagctagt tggcgcgcca
     3121 acctggccgg ccttgataac ttcgtataat gtatgctata cgaagttatc cgaatcgcaa
     3181 taacttcgta taaagtatcc tatacgaagt tatcgaagat catcaagctt atcgataatc
     3241 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     3301 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3361 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3421 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3481 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3541 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3601 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3661 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3721 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3781 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
     3841 tgctcgagag atcgatctgc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3901 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3961 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4021 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggacacgtgc
     4081 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4141 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcggc ctcagtgagc
     4201 gagcgagcgc gcagctgcct gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac
     4261 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4321 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4381 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4441 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4501 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4561 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4621 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4681 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4741 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4801 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4861 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4921 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4981 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5041 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5101 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5161 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5221 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5281 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5341 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5401 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5461 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5521 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5581 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5641 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5701 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5761 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5821 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5881 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5941 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6001 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     6061 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     6121 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     6181 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     6241 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca ta
//
