LOCUS       sequence-329413-        6288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cre-dependent ChR2-TdTomato under control of the TRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6288)
  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 6288)
  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
FEATURES             Location/Qualifiers
     source          1..6288
                     /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             complement(1437..2147)
                     /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(1517..1540)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1715..1734)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1984..2002
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2144..2153
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2163..3089)
                     /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      3086..3095
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3132..3165
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3176..3209
                     /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    3233..3821
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3286..3306)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3704..3715)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3856..3873)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3862..4069
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4092..4221
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4283..4871)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4363..4382)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5042..5833,5834..5902))
                     /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     5665..5684
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5903..6007)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6075..6093
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6131..6153)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6253..6272
                     /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 ttgtacagct cgtccatgcc gccggtggag tggcggccct cggcgcgctc gtactgttcc
     1501 acgatggtgt agtcctcgtt gtgggaggtg atgtccagct tgatgttgac gttgtaggcg
     1561 ccgggcagct gcacgggctt cttggccttg taggtggtct tgacctcggc gtcgtagtgg
     1621 ccgccgtcct tcagcttcag cctctgcttg atctcgccct tcagggcgcc gtcctcgggg
     1681 tacatccgct cggaggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg
     1741 gaggggaagt tggtgccgcg cagcttcacc ttgtagatga actcgccgtc ctgcagggag
     1801 gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg
     1861 aagccctcgg ggaaggacag cttcaagtag tcggggatgt cggcggggtg cttcacgtag
     1921 gccttggagc cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg cagggggccg
     1981 cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg
     2041 ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgtgcacctt gaagcgcatg
     2101 aactccttga tgatggccat gttgtcctcc tcgcccttgc tcaccatggt ggcgaccggt
     2161 gccacggctc cggcctcggc ttcgtcttcg acgagagtct cgacctcgat ctccgttccg
     2221 ccgatgttca gtttggtggt tttgcggata tctccgtgaa tcaatatgtg ctcgtggatc
     2281 aggacgcgca ggtagtgtcc caacaacccc caacaatttt tactcatcag atcaataatc
     2341 gtgtgaccta cggtggagcc atagacgctc aggacgccaa aaccttcggg ccccaaaatg
     2401 aagagaattg ggaacatacc ccagctcacg aaaaacagcc atgccatgcc ggtcacgacc
     2461 tggcggcacc gaccctttgg cacagtatga taaccctcga tatatgcttt ggcggcgtga
     2521 aaaaatgtgt tcgcgccata gcacaatcca agacaaaaga agatgacttt aacatagccg
     2581 gttgccatgg cgctggtagc cccccacacg atagtcccga tgtctgagac aaggagtccc
     2641 atggttctcc tgctgtagtc gttgctcagg ccggtgaggt tgctcaggcg gataaggatg
     2701 acaggacaag tgagcagcca ctctgcatag cgcagccact gcacccggtg tcctgtggca
     2761 aggtagagca tagagggatt cttaaactca aaaaagaact cgagaatcac cttaaccatt
     2821 tcaatggcgc acacatagat ctcctcccag ccgcatgtag atttccaggt ttggtaggca
     2881 tagaacatca gcagcaaaat gctgaatcct gctgcaagcc actgcaggac atttgacgcg
     2941 gtctgagcgc cgttcgtgcc gcgagattca atccatccgg cacagtaaca ttgatcctca
     3001 gggaccagga cggacccgtt caccaccaca ggattagtaa cgaacaaaag ttcgcgtccg
     3061 acggcagaca aagcgccgcc atagtccatg gtggccgcta gctagttggc gcgccaacct
     3121 ggccggcctt gataacttcg tataatgtat gctatacgaa gttatccgaa tcgcaataac
     3181 ttcgtataaa gtatcctata cgaagttatc gaagatcatc aagcttatcg ataatcaacc
     3241 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     3301 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     3361 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3421 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3481 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3541 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3601 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt
     3661 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3721 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3781 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct
     3841 cgagagatcg atctgcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3901 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3961 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4021 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggac acgtgcggac
     4081 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     4141 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggcggcctca gtgagcgagc
     4201 gagcgcgcag ctgcctgcag gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4261 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4321 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4381 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4441 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4501 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4561 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4621 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4681 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4741 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4801 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4861 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4921 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4981 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5041 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5101 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5161 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5221 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5281 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5341 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5401 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5461 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5521 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5581 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5641 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5701 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5761 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5821 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5881 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5941 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6001 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     6061 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     6121 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     6181 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     6241 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccata
//
