LOCUS       Exported                6479 bp ds-DNA     circular SYN 02-AUG-2021
DEFINITION  Cation channelrhodopsin ChroME2s targeted to the neuronal soma and 
            proximal dendrites and separated from nuclear mRuby3 by P2A in a 
            viral vector.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-TRE-ChroME2s-ST-P2A-NLS-mRuby3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6479)
  TITLE     High performance microbial opsins for spatially and temporally 
            precise perturbations of large neuronal networks
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6479)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 2, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6479
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    165..183
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    202..220
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    239..257
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    276..294
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    313..331
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    350..368
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    387..405
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    424..442
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     464..488
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1547..1570
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1781..1837
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1847..1867
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1877..2587
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     misc_feature    2606..3194
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2659..2679)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3077..3088)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3226..3702
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3425..3444)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3742..3882
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3742..3871
                     /label=AAV2 ITR
     rep_origin      3957..4412
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4044..4063)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4254..4275
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4429..4448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4548..4570
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4608..4626)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4694..4798
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4799..5659
                     /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(5017..5036)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5830..6418
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6319..6338
                     /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 gcgtatcaag tgccacctga cgtctcccta tcagtgatag
      181 agaagtcgac acgtctcgag ctccctatca gtgatagaga aggtacgtct agaacgtctc
      241 cctatcagtg atagagaagt cgacacgtct cgagctccct atcagtgata gagaaggtac
      301 gtctagaacg tctccctatc agtgatagag aagtcgacac gtctcgagct ccctatcagt
      361 gatagagaag gtacgtctag aacgtctccc tatcagtgat agagaagtcg acacgtctcg
      421 agctccctat cagtgataga gaagctaccc cctatataag cagagctcgt ttagtgaacc
      481 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc
      541 gatccagcct ggatccggta ccatggaaac agccgccaca atgacccacg cctttatctc
      601 agccgtgcct agcgccgaag ccacaattag aggcctgctg agcgccgcag cagtggtgac
      661 accagcagca gacgctcacg gagaaacctc taacgccaca acagccggag ccgatcacgg
      721 ttgcttcccc cacatcaacc acggaaccga gctgcagcac aagatcgcag tgggactcca
      781 gtggttcacc gtgatcgtgg ctatcgtgca gctcatcttc tacggttggc acagcttcaa
      841 ggccacaacc ggctgggagg aggtctacgt ctgcgtgatc gagctcgtca agtgcttcat
      901 cgagctgttc cacgaggtcg acagcccagc cacagtgtac cagaccaacg gaggagccgt
      961 gatttggctg cggtacagcg agtggctcct gacttgcccc gtgatcctga tccacctgag
     1021 caacctgacc ggactgcacg aagagtacag caagcggacc atgaccatcc tggtgaccga
     1081 catcggcaac atcgtgtggg ggatcacagc cgcctttaca aagggccccc tgaagatcct
     1141 gttcttcatg atcggcctgt tctacggcgt gacttgcttc ttccagatcg ccaaggtgta
     1201 tatcgagagc taccacaccc tgcccaaagg cgtctgccgg aagatttgca agatcatggc
     1261 ctacgtcttc ttctgctctt ggctgatgtt ccccgtgatg ttcatcgccg gacacgaggg
     1321 actgggcctg atcacacctt acaccagcgg aatcggccac ctgatcctgg atctgatcgc
     1381 caagaacact tggggcttcc tgggccacca cctgagagtg aagatccacg agcacatcct
     1441 gatccacggc gacatccgga agacaaccac catcaacgtg gccggcgaga acatggagat
     1501 cgagaccttc gtcgacgagg aggaggaggg aggagtggca ccggtagatt acaaagacga
     1561 tgacgataag cagtcccagc ccatcctcaa caccaaggag atggccccgc agagcaagcc
     1621 tccagaggag ctggagatga gcagcatgcc cagccccgtg gcccctctgc ccgcacgcac
     1681 ggagggcgtc atcgacatgc ggagcatgtc cagcattgac agcttcatca gctgtgccac
     1741 ggacttccct gaagccacca gattcgcggc aggaagcgga gctactaact tcagcctgct
     1801 gaagcaggct ggagacgtgg aggagaaccc tggacctact agtgcaccca agaagaagag
     1861 gaaggtggcg gccgctatgg tgtctaaggg cgaagagctg atcaaggaaa atatgcgtat
     1921 gaaggtggtc atggaaggtt cggtcaacgg ccaccaattc aaatgcacag gtgaaggaga
     1981 aggcagaccg tacgagggag tgcaaaccat gaggatcaaa gtcatcgagg gaggacccct
     2041 gccatttgcc tttgacattc ttgccacgtc gttcatgtat ggcagccgta cctttatcaa
     2101 gtacccggcc gacatccctg atttctttaa acagtccttt cctgagggtt ttacttggga
     2161 aagagttacg agatacgaag atggtggagt cgtcaccgtc acgcaggaca ccagccttga
     2221 ggatggcgag ctcgtctaca acgtcaaggt cagaggggta aactttccct ccaatggtcc
     2281 cgtgatgcag aagaagacca agggttggga gcctaataca gagatgatgt atccagcaga
     2341 tggtggtctg agaggataca ctgacatcgc actgaaagtt gatggtggtg gccatctgca
     2401 ctgcaacttc gtgacaactt acaggtcaaa aaagaccgtc gggaacatca agatgcccgg
     2461 tgtccatgcc gttgatcacc gcctggaaag gatcgaggag agtgacaatg aaacctacgt
     2521 agtgcaaaga gaagtggcag ttgccaaata cagcaacctt ggtggtggca tggacgagct
     2581 gtacaagtga attaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     2641 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     2701 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     2761 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     2821 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     2881 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     2941 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     3001 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc
     3061 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     3121 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     3181 ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt
     3241 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     3301 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     3361 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     3421 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     3481 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     3541 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     3601 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     3661 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     3721 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     3781 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     3841 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     3901 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     3961 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4021 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4081 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4141 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4201 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4261 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     4321 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4381 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4441 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4501 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4561 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4621 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4681 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4741 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4801 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4861 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4921 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4981 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5041 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5101 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5161 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5221 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5281 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5341 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5401 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5461 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5521 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5581 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5641 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5701 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5761 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5821 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5881 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5941 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6001 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6061 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6121 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6181 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6241 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6301 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6361 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6421 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
