LOCUS       Exported                7156 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses Venus-tagged parapinopsin in mammalian cells for 
            photoswitchable control of inhibitory GPCR signaling cascades..
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3-PPO-Venus
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7156)
  AUTHORS   Copits BA, O'Neill PR, Gowrishankar R, Yoo JJ, Meshik X, Parker KE, 
            Spangler SM, Vasquez AM, Elerding AJ, Stander MC, Kalyanaraman V, 
            Vogt SK, Samineni VK, Gautam N, Sunahara RK, Gereau RW, Bruchas MR.
  TITLE     A photoswitchable GPCR-based opsin for presynaptic silencing.
  JOURNAL   BioRxiv
REFERENCE   2  (bases 1 to 7156)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7156
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        89..468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        469..672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     623..643
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     717..736
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        717..735
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1844..1853
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1850..2569
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1895..1916)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2156..2175)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2503..2524
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2601..2618)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2607..2831
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2877..3305
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2964..2983)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3174..3195
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3314..3334)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3319..3648
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3499..3634
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3561..3580
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3715..4509
                     /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(3769..3788)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4379..4398
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4683..4804
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4720..4739)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4774..4793
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4853..4869)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4853..4869)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4866..4888)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4877..4893
                     /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(4901..4931)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4946..4967
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5084..5101)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5255..5843)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5335..5354)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6014..6874)
                     /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     6637..6656
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6875..6979)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7054..7073)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
       61 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      661 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
      721 acgactcact atagggagac ccaagctggc tagcgtttaa acttaagctt ggtaccgagc
      781 tcggatccac tagtccagtg tggtggaatt catggagaac ttgacctcgc tcgacctcct
      841 gcccaacggc gaggtcccgt tgatgccccg ctacggcttc accatcctcg ccgtgatcat
      901 ggccgtgttc accatcgcct cgctcgtgct caacagcacc gtcgtcatcg tcaccctgcg
      961 ccaccgccag ctacgccacc cgctcaactt ctcgctcgtc aacctcgccg tggcggacct
     1021 gggcgtcacg gtgttcggcg ccagcctcgt cgtggagacc aacgccgtcg ggtacttcaa
     1081 cctcggccgc gtcggctgcg tcatcgaagg gttcgccgtc gctttcttcg gcatcgccgc
     1141 tctgtgcacg atcgccgtga tcgccgtcga tcgcttcgtg gtggtgtgca agccgctggg
     1201 cacgctgatg ttcacgcggc gccacgcgct gctgggcatc gcctgggcct ggctctggtc
     1261 gttcgtgtgg aacacgccgc cgctcttcgg ctggggcagc tacgagctgg agggcgtgcg
     1321 gacgtcgtgc gcgcccgact ggtacagccg cgaccccgcc aacgtgtcgt acattacgag
     1381 ctacttcgcc ttctgcttcg ccatcccctt cctcgtcatc gtggtggcgt acggccgcct
     1441 catgtggacc ctccaccagg tggccaagct ggggatgggc gagagcggca gcaccgccaa
     1501 ggcggaggcg caggtgtcgc gcatggtggt ggtcatggtg gtggccttcc tcgtctgctg
     1561 gctgccctac gcgctcttcg ccatgatcgt ggtgaccaag cccgacgtgt acatcgaccc
     1621 ggtcatcgcc acactgccca tgtacctgac caagacgagc acggtctaca accccatcat
     1681 ctacatcttc atgaaccgcc agttccggga ctgcgccgtg cccttcctgc tctgcggacg
     1741 caacccctgg gcagagcctt cctcggagtc ggcgacggcg gcctccacgt cggccaccag
     1801 cgtgaccctg gcgtccgcgc cggggcaggt ctccccgagc gcggccgcca tggtgagcaa
     1861 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1921 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1981 cctgaagctg atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     2041 cctgggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     2101 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2161 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2221 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2281 caactacaac agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc
     2341 caacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca
     2401 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta
     2461 ccagtccaaa ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     2521 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaat ctagagggcc
     2581 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     2641 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2701 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     2761 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     2821 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     2881 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2941 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3001 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3061 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3121 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3181 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3241 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3301 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3361 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3421 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3481 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3541 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     3601 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     3661 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     3721 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3781 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3841 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     3901 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     3961 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4021 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4081 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4141 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4201 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4261 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4321 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4381 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4441 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4501 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4561 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4621 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4681 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4741 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4801 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4861 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4921 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4981 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5041 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5101 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5161 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5221 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5281 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5341 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5401 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5461 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5521 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5581 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5641 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5701 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5761 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5821 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5881 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5941 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6001 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6061 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6121 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6181 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6241 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6301 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6361 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6421 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6481 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6541 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6601 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6661 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6721 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6781 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6841 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6901 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6961 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     7021 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     7081 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     7141 ttaagctaca acaagg
//
