LOCUS       sequence-181303-        6211 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Reporter construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6211)
  AUTHORS   Wang W, Wildes CP, Pattarabanjird T, Sanchez MI, Glober GF, Matthews
            GA, Tye KM, Ting AY
  TITLE     A light- and calcium-gated transcription factor for imaging and 
            manipulating activated neurons.
  JOURNAL   Nat Biotechnol. 2017 Jun 26. doi: 10.1038/nbt.3909.
  PUBMED    28650461
REFERENCE   2  (bases 1 to 6211)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2017 Jun 26. doi: 10.1038/nbt.3909."
COMMENT     Sequence Label: TRE-ChrimsonR-mCherry
FEATURES             Location/Qualifiers
     source          1..6211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    77..95
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    112..130
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    148..166
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    184..202
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    219..237
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    255..273
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        285..323
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     320..344
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      372..381
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             378..1427
                     /codon_start=1
                     /product="red light-drivable channelrhodopsin from 
                     Chlamydomonas noctigama (Klapoetke et al., 2014)"
                     /label=ChrimsonR
                     /note="K176R mutant with accelerated closing kinetics"
                     /translation="MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTE
                     WWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSI
                     AIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRY
                     FEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIV
                     SCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLK
                     LSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEE
                     FVEEEDEDTV"
     regulatory      1491..1500
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1497..2207
                     /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(1642..1660)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1910..1929
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2239..2827
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2292..2312)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2710..2721)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2859..3335
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3058..3077)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3375..3515
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3375..3504
                     /label=AAV2 ITR
     rep_origin      3590..4045
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3677..3696)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3887..3908
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4062..4081)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4181..4203
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4241..4259)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4327..4431
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4432..4500,4501..5292)
                     /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(4650..4669)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5463..6051
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5952..5971
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6113..6211,1..31)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ggagtggcca actccatcac taggggttcc tgcggccgca cgcgtccttc aggtccgagg
       61 ttctagacga gtttactccc tatcagtgat agagaacgat gtcgagttta ctccctatca
      121 gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt
      181 tactccctat cagtgataga gaacgtatgt cgagtttatc cctatcagtg atagagaacg
      241 tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgaggtagg cgtgtacggt
      301 gggaggccta tataagcaga gctcgtttag tgaaccgtca gatcgcaaag ggcgaattcg
      361 atccaccggt cgccaccatg gctgagctga tcagcagcgc caccagatct ctgtttgccg
      421 ccggaggcat caacccttgg cctaacccct accaccacga ggacatgggc tgtggaggaa
      481 tgacacctac aggcgagtgc ttcagcaccg agtggtggtg tgacccttct tacggactga
      541 gcgacgccgg atacggatat tgcttcgtgg aggccacagg cggctacctg gtcgtgggag
      601 tggagaagaa gcaggcttgg ctgcacagca gaggcacacc aggagaaaag atcggcgccc
      661 aggtctgcca gtggattgct ttcagcatcg ccatcgccct gctgacattc tacggcttca
      721 gcgcctggaa ggccacttgc ggttgggagg aggtctacgt ctgttgcgtc gaggtgctgt
      781 tcgtgaccct ggagatcttc aaggagttca gcagccccgc cacagtgtac ctgtctaccg
      841 gcaaccacgc ctattgcctg cgctacttcg agtggctgct gtcttgcccc gtgatcctga
      901 tcagactgag caacctgagc ggcctgaaga acgactacag caagcggacc atgggcctga
      961 tcgtgtcttg cgtgggaatg atcgtgttcg gcatggccgc aggactggct accgattggc
     1021 tcaagtggct gctgtatatc gtgtcttgca tctacggcgg ctacatgtac ttccaggccg
     1081 ccaagtgcta cgtggaagcc aaccacagcg tgcctaaagg ccattgccgc atggtcgtga
     1141 agctgatggc ctacgcttac ttcgcctctt ggggcagcta cccaatcctc tgggcagtgg
     1201 gaccagaagg actgctgaag ctgagccctt acgccaacag catcggccac agcatctgcg
     1261 acatcatcgc caaggagttt tggaccttcc tggcccacca cctgaggatc aagatccacg
     1321 agcacatcct gatccacggc gacatccgga agaccaccaa gatggagatc ggaggcgagg
     1381 aggtggaagt ggaagagttc gtggaggagg aggacgagga cacagtggcg gcaccggtag
     1441 tagcagtgag cggtggaagc ggttctggaa gtgctcggga tccaccggtc gccaccatgg
     1501 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     1561 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1621 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
     1681 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     1741 ccgacatccc cgactacttg aagctgtcct tccccgaggg attcaagtgg gagcgcgtga
     1801 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     1861 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     1921 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     1981 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     2041 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     2101 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg
     2161 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaaact agtaccggta
     2221 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     2281 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     2341 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     2401 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     2461 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     2521 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     2581 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     2641 tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc
     2701 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     2761 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     2821 tccccgcatc gataccgagc gctgctcgag agatctacgg gtggcatccc tgtgacccct
     2881 ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa
     2941 taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga
     3001 ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct
     3061 attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct
     3121 gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga
     3181 ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc
     3241 tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat
     3301 tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta accacgtgcg
     3361 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     3421 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     3481 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     3541 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     3601 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3661 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3721 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3781 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     3841 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3901 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     3961 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4021 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     4081 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4141 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4201 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4261 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4321 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4381 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4441 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4501 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4561 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4621 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4681 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4741 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4801 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4861 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4921 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4981 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5041 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5101 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5161 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5221 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5281 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5341 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5401 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5461 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5521 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5581 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5641 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5701 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5761 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5821 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5881 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5941 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6001 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6061 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg
     6121 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc
     6181 ggcctcagtg agcgagcgag cgcgcagaga g
//
