LOCUS       sequence-288142-        7342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mScarlet-tagged parapinopsin in mammalian cells for 
            photoswitchable control of inhibitory GPCR signaling cascades..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7342)
  AUTHORS   Copits BA, Gowrishankar R, O'Neill PR, Li JN, Girven KS, Yoo JJ, 
            Meshik X, Parker KE, Spangler SM, Elerding AJ, Brown BJ, Shirley SE,
            Ma KKL, Vasquez AM, Stander MC, Kalyanaraman V, Vogt SK, Samineni 
            VK, Patriarchi T, Tian L, Gautam N, Sunahara RK, Gereau RW 4th, 
            Bruchas MR
  TITLE     A photoswitchable GPCR-based opsin for presynaptic inhibition.
  JOURNAL   Neuron. 2021 Jun 2;109(11):1791-1809.e11. doi: 
            10.1016/j.neuron.2021.04.026. Epub 2021 May 11.
  PUBMED    33979635
REFERENCE   2  (bases 1 to 7342)
  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"; doi: 
            "10.1016/j.neuron.2021.04.026"; journalName: "Neuron"; date: 
            "2021-06-2- 2"; volume: "109"; issue: "11"; pages: "1791-1809.e11"
COMMENT     Sequence Label: pcDNA3-PPO-mScarlet
FEATURES             Location/Qualifiers
     source          1..7342
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        57..436
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        437..640
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     591..611
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     685..704
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        685..703
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             919..948
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1990..2685
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2123..2141)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2391..2410
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(2717..2735)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2718..2735)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2755..2772)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2761..2985
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3031..3459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3118..3137)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3328..3349
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3468..3488)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3473..3802
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3653..3788
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3715..3734
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3869..4663
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3923..3942)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4533..4552
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4837..4958
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4874..4893)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4928..4947
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5007..5023)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5007..5023)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5020..5042)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5031..5047
                     /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(5055..5061,5062..5079,5080..5085))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5100..5121
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5238..5255)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5409..5997)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5489..5508)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6168..6959,6960..7028))
                     /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     6791..6810
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7029..7133)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7208..7227)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
       61 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      121 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      181 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      241 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      301 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      361 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      421 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      481 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      541 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      601 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      661 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagcttggta
      721 ccgagctcgg atcccggtcc gaagcgcgcg gaattcagat ctggtaccac gcgtatcgat
      781 aagcttgatt cgagctcgcc ccgcccccag ccagtgcgct tacctgccag actgcgcgcc
      841 atgaagacga tcatcgccct gagctacatc ttctgcctgg tgttcgccga ctacaaggac
      901 gatgatgacg ccatgagcca tcaccatcac catcaccatc accatcacgc catggagaac
      961 ttgacctcgc tcgacctcct gcccaacggc gaggtcccgt tgatgccccg ctacggcttc
     1021 accatcctcg ccgtgatcat ggccgtgttc accatcgcct cgctcgtgct caacagcacc
     1081 gtcgtcatcg tcaccctgcg ccaccgccag ctacgccacc cgctcaactt ctcgctcgtc
     1141 aacctcgccg tggcggacct gggcgtcacg gtgttcggcg ccagcctcgt cgtggagacc
     1201 aacgccgtcg ggtacttcaa cctcggccgc gtcggctgcg tcatcgaagg gttcgccgtc
     1261 gctttcttcg gcatcgccgc tctgtgcacg atcgccgtga tcgccgtcga tcgcttcgtg
     1321 gtggtgtgca agccgctggg cacgctgatg ttcacgcggc gccacgcgct gctgggcatc
     1381 gcctgggcct ggctctggtc gttcgtgtgg aacacgccgc cgctcttcgg ctggggcagc
     1441 tacgagctgg agggcgtgcg gacgtcgtgc gcgcccgact ggtacagccg cgaccccgcc
     1501 aacgtgtcgt acattacgag ctacttcgcc ttctgcttcg ccatcccctt cctcgtcatc
     1561 gtggtggcgt acggccgcct catgtggacc ctccaccagg tggccaagct ggggatgggc
     1621 gagagcggca gcaccgccaa ggcggaggcg caggtgtcgc gcatggtggt ggtcatggtg
     1681 gtggccttcc tcgtctgctg gctgccctac gcgctcttcg ccatgatcgt ggtgaccaag
     1741 cccgacgtgt acatcgaccc ggtcatcgcc acactgccca tgtacctgac caagacgagc
     1801 acggtctaca accccatcat ctacatcttc atgaaccgcc agttccggga ctgcgccgtg
     1861 cccttcctgc tctgcggacg caacccctgg gcagagcctt cctcggagtc ggcgacggcg
     1921 gcctccacgt cggccaccag cgtgaccctg gcgtccgcgc cggggcaggt ctccccgagc
     1981 ggcggcagca tggtgagcaa gggcgaggca gtgatcaagg agttcatgcg gttcaaggtg
     2041 cacatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     2101 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     2161 tcctgggaca tcctgtcccc tcagttcatg tacggctcca gggccttcac caagcacccc
     2221 gccgacatcc ccgactacta taagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg
     2281 atgaacttcg aggacggcgg cgccgtgacc gtgacccagg acacctccct ggaggacggc
     2341 accctgatct acaaggtgaa gctccgcggc accaacttcc ctcctgacgg ccccgtaatg
     2401 cagaagaaga caatgggctg ggaagcgtcc accgagcggt tgtaccccga ggacggcgtg
     2461 ctgaagggcg acattaagat ggccctgcgc ctgaaggacg gcggccgcta cctggcggac
     2521 ttcaagacca cctacaaggc caagaagccc gtgcagatgc ccggcgccta caacgtcgac
     2581 cgcaagttgg acatcacctc ccacaacgag gactacaccg tggtggaaca gtacgaacgc
     2641 tccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaaac tcgagcatgc
     2701 atctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga
     2761 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2821 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     2881 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     2941 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3001 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3061 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3121 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3181 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3241 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3301 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3361 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     3421 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     3481 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3541 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3601 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3661 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3721 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     3781 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     3841 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     3901 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     3961 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4021 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     4081 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     4141 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     4201 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     4261 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     4321 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     4381 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     4441 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     4501 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4561 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4621 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     4681 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     4741 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4801 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4861 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4921 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4981 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5041 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5101 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5161 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5221 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5281 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5341 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5401 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5461 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5521 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5581 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5641 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5701 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5761 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5821 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5881 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5941 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6001 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6061 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6121 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6181 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6241 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6301 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6361 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6421 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6481 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6541 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6601 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6661 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6721 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6781 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6841 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6901 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6961 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7021 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7081 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7141 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     7201 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     7261 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     7321 ccgacaattg catgaagaat ct
//
