LOCUS       sequence-361777-        6167 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of CheRiff channelrhodopsin linked to mKate2 from a 
            Tet-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6167)
  TITLE     cFos ensembles in the dentate gyrus rapidly segregate over time and 
            do not form a stable map of space
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6167)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV TRE-Tight CheRiff-mKate2
FEATURES             Location/Qualifiers
     source          1..6167
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    12..30
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    48..66
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    83..101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    119..137
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    155..173
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    190..208
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    226..244
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        256..294
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     291..315
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             340..1287
                     /codon_start=1
                     /product="blue light-activated optogenetic actuator derived
                     from Scherffelia dubia channelrhodopsin (Hochbaum et al., 
                     2014)"
                     /label=CheRiff
                     /translation="MGGAPAPDAHSAPPGNDSAGGSEYHAPAGYQVNPPYHPVHGYEEQ
                     CSSIYIYYGALWEQETARGFQWFAVFLSALFLAFYGWHAYKASVGWEEVYVCSVELIKV
                     ILEIYFEFTSPAMLFLYGGNITPWLRYAEWLLTCPVILIHLSNITGLSEAYNKRTMALL
                     VSDLGTICMGVTAALATGWVKWLFYCIGLVYGTQTFYNAGIIYVESYYIMPAGGCKKLV
                     LAMTAVYYSSWLMFPGLFIFGPEGMHTLSVAGSTIGHTIADLLSKNIWGLLGHFLRIKI
                     HEHIIMYGDIRRPVSSQFLGRKVDVLAFVTEEDKV"
     misc_feature    2101..2689
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2154..2174)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2572..2583)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2692..2708)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2693..2709)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    2776..3252
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2975..2994)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3292..3432
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3292..3421
                     /label=AAV2 ITR
     rep_origin      3507..3962
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3594..3613)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3804..3825
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3979..3998)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4098..4120
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4158..4176)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4244..4348
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4349..4417,4418..5209)
                     /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(4567..4586)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5380..5968
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5869..5888
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6030..6159
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 acgcgtttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
       61 tagagaacga tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc
      121 cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg
      181 tcgagtttat ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
      241 gagaacgtat gtcgaggtag gcgtgtacgg tgggaggcct atataagcag agctcgttta
      301 gtgaaccgtc agatcgcctg gagaattcaa gctgctagca tgggcggagc tcctgctcca
      361 gacgctcaca gcgccccacc tggaaacgat tctgccggag gcagtgagta ccatgcccca
      421 gctggatatc aagtgaatcc accctaccac cccgtgcatg ggtatgagga acagtgcagc
      481 tccatctaca tctactatgg ggccctgtgg gagcaggaaa cagctagggg cttccagtgg
      541 tttgccgtgt tcctgtctgc cctgtttctg gctttctacg gctggcacgc ctataaggcc
      601 agcgtgggat gggaggaagt gtacgtgtgc tccgtggagc tgatcaaagt gattctggag
      661 atctatttcg agttcaccag tcctgctatg ctgttcctgt acggagggaa cattacccca
      721 tggctgagat atgccgaatg gctgctgaca tgtcccgtga tcctgattca tctgtctaac
      781 atcaccggcc tgagtgaggc atacaataag cggacaatgg ctctgctggt gtccgacctg
      841 ggaactattt gcatgggagt gacagccgct ctggccactg ggtgggtgaa gtggctgttt
      901 tactgtatcg gcctggtgta tggaacccag acattctaca acgctggaat catctacgtg
      961 gagtcttact atatcatgcc tgccggcggc tgtaagaaac tggtgctggc catgactgcc
     1021 gtgtactatt ctagttggct gatgtttccc ggcctgttca tctttgggcc tgaaggcatg
     1081 cacaccctga gcgtggctgg gtccactatt ggccatacca tcgccgacct gctgtccaag
     1141 aatatttggg gactgctggg gcacttcctg cggatcaaaa ttcacgagca tatcattatg
     1201 tacggcgata tcaggagacc agtgagctcc cagtttctgg gacgcaaggt ggacgtgctg
     1261 gccttcgtga cagaggaaga taaagtggcg gccgccaaga gcaggatcac cagcgagggc
     1321 gagtacatcc ccctggacca gatcgacatc aacgtgggac ccgtggctgc tcctgtggtg
     1381 gctcatatgg tgtccgagct gatcaaagaa aacatgcaca tgaagctgta catggaaggc
     1441 accgtgaaca accaccactt caagtgcacc agcgagggcg agggcaagcc ttacgagggc
     1501 acccagacca tgagaatcaa ggccgtggaa ggcggccctc tgcccttcgc ctttgacatc
     1561 ctggccacca gctttatgta cggcagcaag accttcatca accacaccca gggcatcccc
     1621 gatttcttca agcagagctt ccccgagggc ttcacctggg agagagtgac cacatacgag
     1681 gacggcggag tgctgaccgc cacccaggat acaagcctgc aggacggctg cctgatctac
     1741 aacgtgaaga tcagaggcgt gaacttcccc agcaacggcc ctgtgatgca gaagaaaaca
     1801 ctgggctggg aggccagcac cgagacactg tatcctgctg acggcggcct ggaaggcaga
     1861 gccgatatgg ccctgaaact cgtgggcgga ggccacctga tctgtaacct gaaaaccacc
     1921 tacaggtcca agaagcccgc caagaacctg aagatgcccg gcgtgtacta cgtggacaga
     1981 aggctggaaa gaatcaaaga ggccgacaaa gaaacctacg tggaacagca cgaggtggcc
     2041 gtggctagat actgcgacct gcctagcaag ctgggacaca gatgacaaaa gcttatcgat
     2101 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     2161 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     2221 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     2281 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     2341 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     2401 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     2461 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     2521 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     2581 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     2641 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt
     2701 cgacctcgag acgcgatcgc ggcgcgccat tcgatggcct ctctggcctc gacctcgagt
     2761 ctagagatga tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg
     2821 gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg
     2881 tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg
     2941 caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca
     3001 gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct
     3061 cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt
     3121 ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag
     3181 gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc
     3241 ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc
     3301 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     3361 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     3421 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3481 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     3541 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     3601 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3661 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3721 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3781 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     3841 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     3901 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     3961 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     4021 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4081 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4141 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4201 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4261 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4321 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4381 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4441 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4501 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4561 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4621 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4681 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4741 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4801 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4861 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     4921 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4981 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5041 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     5101 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5161 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5221 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5281 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5341 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5401 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5461 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5521 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5581 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5641 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5701 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5761 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5821 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5881 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5941 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6001 ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac
     6061 tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
     6121 gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgc
//
