LOCUS       sequence-468901-        7546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG-driven, Flp-dependent ChRmine for optogenetic excitation of 
            neurons with an oScarlet tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7546)
  AUTHORS   Falasconi A, Kanodia H, Arber S
  TITLE     Dynamic basal ganglia output signals license and suppress forelimb 
            movements.
  JOURNAL   Nature. 2025 May 28. doi: 10.1038/s41586-025-09066-z.
  PUBMED    40437098
REFERENCE   2  (bases 1 to 7546)
  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: "Nature. 
            2025 May 28. doi: 10.1038/s41586-025-09066-z."
COMMENT     Sequence Label: 
            pAAV-pCAG-FRT(F3F5)-ChRmine3.3-p2A-oScarlet,rev.-WPRE
FEATURES             Location/Qualifiers
     source          1..7546
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        223..526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        528..805
                     /label=chicken beta-actin promoter
     intron          806..1822
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1746..1768
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1830..1849
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1926..1959
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     CDS             complement(2045..2740)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWDRVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2320..2339)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2589..2607
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2741..2797)
                     /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"
     regulatory      3808..3817
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3849..3882)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     misc_feature    3950..4538
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4003..4023)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4421..4432)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4573..4590)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4579..4786
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4809..4949
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4809..4938
                     /label=AAV2 ITR
     rep_origin      5024..5479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5111..5130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5321..5342
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5496..5515)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5615..5637
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5675..5693)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5761..5865
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5866..5934,5935..6726)
                     /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(6084..6103)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6897..7485
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7386..7405
                     /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 gcgaaacaat tctgcaggaa tctagttatt aatagtaatc
      181 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      241 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      301 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      361 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      421 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      481 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      541 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      601 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggg gcgcgccggg
      661 gggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa
      721 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      781 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
      841 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
      901 gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct
      961 tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg
     1021 ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc
     1081 cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag
     1141 tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg
     1201 gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc
     1261 ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc
     1321 gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg
     1381 gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg
     1441 gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct
     1501 tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc
     1561 gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc
     1621 cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct
     1681 ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag
     1741 ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca
     1801 tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca
     1861 ttttggcaaa gaattgattt gataccgcgg gccccgcgtg gacggtaccc ttcaggtcga
     1921 cgtcggaagt tcctattctt caaatagtat aggaacttcc cgaatcgcag aagttcctat
     1981 tcttcaaaag gtataggaac ttcttggcgc gccggaattc ttacacctcg ttctcgtagc
     2041 agaacttgta cagctcgtcc atgccgccgg tggagtggcg gccctcggag cgttcgtact
     2101 gttccaccac ggtgtagtcc tcgttgtggg aggtgatgtc caacttgcgg tccacgttgt
     2161 aggcgccggg catctgcacg ggcttcttgg ccttgtaggt ggtcttgaag tccgccaggt
     2221 agcgcccgcc gtccttcagg cgcagggcca tcttaatgtc gcccttcagc acgccgtcct
     2281 cggggtacaa ccgctcggtg gacgcttccc agcccattgt cttcttctgc attacggggc
     2341 cgtcaggagg gaagttggtg ccgcggagct tcaccttgta gatcagggtg ccgtcctcca
     2401 gggaggtgtc ctgggtcacg gtcacggcgc cgccgtcctc gaagttcatc acgcggtccc
     2461 acttgaagcc ctcggggaag gactgcttat agtagtcggg gatgtcggcg gggtgcttgg
     2521 tgaaggccct ggagccgtac atgaactgag gggacaggat gtcccaggag aagggcaggg
     2581 ggccaccctt ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc
     2641 cctcgccctc gatctcgaac tcgtggccgt tcatggagcc ctccatgtgc accttgaacc
     2701 gcatgaactc cttgatcact gcctcgccct tgctcaccat gggaccgggg ttttcttcca
     2761 cgtctcctgc ttgctttaac agagagaagt tcgtggctcc ggagccgggc gcgcccacgt
     2821 tgatgtcgat ctggtccagg gggatgtact cgccctcgct ggtgatcctg ctcttggcgg
     2881 ccgcagacag tctccgcagg cgctcgccat tgctgctcag cctcatatcc agctccagct
     2941 tctggaactc atcctcgtcg tgtcccagct gctccagcag ccgaaacagc tcctcatcat
     3001 aggtctttct gaaccgggcc agggcaaagc cgtaacagga cttggccacg atgtcgcaca
     3061 cgcagtgggc cacctctgtc cagttatcgt cgatccagcc gaagcctcta gggcagatgg
     3121 cccacaccag gggaaacaga atccacagca tggagaaggt aaacacggcg aacttcagca
     3181 cgtgcaggct gtgctcggcg cctgtatcct gggccagctg ggccagccgg ctatactgct
     3241 tggccacgat gctcatcacg atagagtagg cgaagataaa ccagaaacag ccaaagccat
     3301 accaggcgta ggcgccattc ctcagcctag ggtcgccctc tgcgtagaac tcggccagca
     3361 cgccagacat caggatggca aagatgatgg cgctacaaga cacgcgatag ggggccctca
     3421 gctgatacag cagatcgtac accagcattg ggcaggtcag catgtagtcg gcatacctgt
     3481 aattgccgaa gccgatgatg aaggggccca catcgccgaa gttgtggccc agtgtgtcgg
     3541 ccacgatctt gccgaagtag ctcagcatgg caaagaaatt cacaaacagg gccaggttct
     3601 gctcctcctg atagccggcg taaaaggagg ttctcttgct agggccccga gatgtccagg
     3661 actcatagcc caggtagaat gtggtgattg tcagcacgat aaagcaagac cagtgggcgc
     3721 cgatggccac ggagttcagc ttggtatcca gataccagcc gtccattgtg cccacgtaga
     3781 acatctggtc ggtgcctggt gcgtgtgcca tggtggcgga tcccggcgcg ccaacctggc
     3841 cggccttgga agttcctata ctatttgaag aataggaact tcccgaatcg cagaagttcc
     3901 tatacctttt gaagaatagg aacttccgaa gatcatcaag cttatcgata atcaacctct
     3961 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     4021 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     4081 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     4141 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     4201 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     4261 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     4321 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc
     4381 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     4441 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     4501 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga
     4561 gagatcgatc tgcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     4621 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     4681 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     4741 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggacacg tgcggaccga
     4801 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4861 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4921 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     4981 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     5041 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5101 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5161 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5221 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5281 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5341 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5401 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5461 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5521 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5581 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5641 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     5701 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5761 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5821 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5881 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5941 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     6001 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6061 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6121 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6181 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6241 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6301 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6361 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6421 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6481 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6541 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6601 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6661 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6721 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6781 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6841 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6901 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6961 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7021 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     7081 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7141 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7201 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7261 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7321 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7381 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7441 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7501 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
