LOCUS       sequence-203778-        7792 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Cation channelrhodopsin Chronos fused to mRuby2 fluorophore and 
            targeted to the neuronal soma and proximal dendrites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7792)
  AUTHORS   Pegard NC, Mardinly AR, Oldenburg IA, Sridharan S, Waller L, Adesnik
            H
  TITLE     Three-dimensional scanless holographic optogenetics with temporal 
            focusing (3D-SHOT).
  JOURNAL   Nat Commun. 2017 Oct 31;8(1):1228. doi: 10.1038/s41467-017-01031-3.
  PUBMED    29089483
REFERENCE   2  (bases 1 to 7792)
  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 
            Commun."; date: "2017-10-31"; volume: "8(1):1228. doi"; pages: " 
            10.1038/s41467-017-01031-3"
COMMENT     Sequence Label: pCAGGS-Chronos-mRuby2-ST
FEATURES             Location/Qualifiers
     source          1..7792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          169..1185
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1109..1131
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1193..1212
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2263..2973
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     intron          3197..3329
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3312..3331
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3338..3926
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3391..3411)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3809..3820)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3953..3970)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3959..4070
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4166..4186)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4166..4184)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4205..4221)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4323..4342)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4388..4443
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4442..4461)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4804..4820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4828..4844
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4852..4858,4859..4876,4877..4882))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4897..4918
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4977..5172
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5023..5158
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5178..5312
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5215..5234)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5269..5288
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5380..5397)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5551..6139)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5631..5650)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6310..7101,7102..7170))
                     /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     6933..6952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7171..7275)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        7306..7685
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(7687..7792,1..168)
                     /label=chicken beta-actin promoter
ORIGIN
        1 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
       61 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
      121 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
      181 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
      241 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
      301 tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg
      361 ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc
      421 gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc
      481 gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg
      541 cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg
      601 tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga
      661 gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc
      721 gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg
      781 gccggggagg gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc
      841 gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct
      901 ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg
      961 cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg
     1021 tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc
     1081 tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct
     1141 agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg
     1201 ctggttattg tgctgtctca tcattttggc aaagaattcg gtaccgcggg cccgggatcc
     1261 accggtgcta gtatggaaac agccgccaca atgacccacg cctttatctc agccgtgcct
     1321 agcgccgaag ccacaattag aggcctgctg agcgccgcag cagtggtgac accagcagca
     1381 gacgctcacg gagaaacctc taacgccaca acagccggag ccgatcacgg ttgcttcccc
     1441 cacatcaacc acggaaccga gctgcagcac aagatcgcag tgggactcca gtggttcacc
     1501 gtgatcgtgg ctatcgtgca gctcatcttc tacggttggc acagcttcaa ggccacaacc
     1561 ggctgggagg aggtctacgt ctgcgtgatc gagctcgtca agtgcttcat cgagctgttc
     1621 cacgaggtcg acagcccagc cacagtgtac cagaccaacg gaggagccgt gatttggctg
     1681 cggtacagca tgtggctcct gacttgcccc gtgatcctga tccacctgag caacctgacc
     1741 ggactgcacg aagagtacag caagcggacc atgaccatcc tggtgaccga catcggcaac
     1801 atcgtgtggg ggatcacagc cgcctttaca aagggccccc tgaagatcct gttcttcatg
     1861 atcggcctgt tctacggcgt gacttgcttc ttccagatcg ccaaggtgta tatcgagagc
     1921 taccacaccc tgcccaaagg cgtctgccgg aagatttgca agatcatggc ctacgtcttc
     1981 ttctgctctt ggctgatgtt ccccgtgatg ttcatcgccg gacacgaggg actgggcctg
     2041 atcacacctt acaccagcgg aatcggccac ctgatcctgg atctgatcag caagaacact
     2101 tggggcttcc tgggccacca cctgagagtg aagatccacg agcacatcct gatccacggc
     2161 gacatccgga agacaaccac catcaacgtg gccggcgaga acatggagat cgagaccttc
     2221 gtcgacgagg aggaggaggg aggagtggcg gccgcaccgg taatggtgtc taagggcgaa
     2281 gagctgatca aggaaaatat gcgtatgaag gtggtcatgg aaggttcggt caacggccac
     2341 caattcaaat gcacaggtga aggagaaggc aatccgtaca tgggaactca aaccatgagg
     2401 atcaaagtca tcgagggagg acccctgcca tttgcctttg acattcttgc cacgtcgttc
     2461 atgtatggca gccgtacttt tatcaagtac ccgaaaggca ttcctgattt ctttaaacag
     2521 tcctttcctg agggttttac ttgggaaaga gttacgagat acgaagatgg tggagtcgtc
     2581 accgtcatgc aggacaccag ccttgaggat ggctgtctcg tttaccacgt ccaagtcaga
     2641 ggggtaaact ttccctccaa tggtcccgtg atgcagaaga agaccaaggg ttgggagcct
     2701 aatacagaga tgatgtatcc agcagatggt ggtctgaggg gatacactca tatggcactg
     2761 aaagttgatg gtggtggcca tctgtcttgc tctttcgtaa caacttacag gtcaaaaaag
     2821 accgtcggga acatcaagat gcccggtatc catgccgttg atcaccgcct ggaaaggtta
     2881 gaggaaagtg acaatgaaat gttcgtagta caacgcgaac acgcagttgc caagttcgcc
     2941 gggcttggtg gtgggatgga cgagctgtac aaggccctag agcagtccca gcccatcctc
     3001 aacaccaagg agatggcccc gcagagcaag cctccagagg agctggagat gagcagcatg
     3061 cccagccccg tggcccctct gcccgctcgc acggagggcg tcatcgacat gcggagcatg
     3121 tccagcattg acagcttcat cagctgtgcc acggacttcc ctgaagccac cagattctaa
     3181 ggactcgagg ccgcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
     3241 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
     3301 tgacatccac tttgcctttc tctccacagg tgtcgacaat caacctctgg attacaaaat
     3361 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3421 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3481 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     3541 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     3601 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3661 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3721 gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct
     3781 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     3841 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     3901 gatctccctt tgggccgcct ccccgcctgg aattcgagct cggtacgatc agcctcgact
     3961 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4021 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4081 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4141 gaagacaata gcccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     4201 catggtcata gctgtttcct gtgtgaaatt gttatccgct aattcactcc tcaggtgcag
     4261 gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga
     4321 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     4381 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     4441 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     4501 tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga
     4561 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg
     4621 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta
     4681 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc
     4741 atagctgtcc ctcttctctt atggagatcc ctcgacctgc agcccaagct tggcgtaatc
     4801 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4861 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4921 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat
     4981 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     5041 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     5101 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     5161 ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5221 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5281 aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc
     5341 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5401 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5461 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5521 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5581 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5641 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5701 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5761 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5821 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5881 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5941 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6001 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6061 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6121 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6181 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6241 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6301 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6361 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6421 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6481 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6541 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6601 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6661 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6721 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6781 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6841 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6901 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6961 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7021 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7081 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7141 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7201 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7261 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggtcgacat tgattattga
     7321 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     7381 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     7441 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     7501 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     7561 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     7621 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     7681 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
     7741 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gc
//
