LOCUS       sequence-209625-        6541 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of a chimeric G-protein coupled receptor 
            (Rhodopsin-GPR32; Opto-GPR32; B5).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6541)
  AUTHORS   Morri M, Sanchez-Romero I, Tichy AM, Kainrath S, Gerrard EJ, 
            Hirschfeld PP, Schwarz J, Janovjak H
  TITLE     Optical functionalization of human Class A orphan G-protein-coupled 
            receptors.
  JOURNAL   Nat Commun. 2018 May 16;9(1):1950. doi: 10.1038/s41467-018-04342-1.
  PUBMED    29769519
REFERENCE   2  (bases 1 to 6541)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-05-16"; volume: "9(1):1950. doi"; pages: " 
            10.1038/s41467-018-04342-1"
COMMENT     Sequence Label: Opto-GPR32
FEATURES             Location/Qualifiers
     source          1..6541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        454..657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     608..628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     702..721
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        702..720
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             821..853
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     CDS             878..916
                     /codon_start=1
                     /product="segment of bovine rhodopsin containing a signal 
                     for N-linked glycosylation (Favaloro et al., 2008)"
                     /label=opsin tag
                     /note="The asparagine in the NKT tripeptide will be 
                     glycosylated if this sequence is exposed to the ER lumen."
                     /translation="GPNFYVPFSNKTG"
     primer_bind     complement(1971..1988)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1977..2201
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2247..2675
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2334..2353)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2544..2565
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2684..2704)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2689..3018
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2869..3004
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2931..2950
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3085..3879
                     /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(3139..3158)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3749..3768
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4053..4174
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4090..4109)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4144..4163
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4223..4239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4223..4239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4236..4258)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4247..4263
                     /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(4271..4277,4278..4295,4296..4301))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4316..4337
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4454..4471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4625..5213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4705..4724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5384..6175,6176..6244))
                     /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     6007..6026
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6245..6349)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6424..6443)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
       61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      661 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
      721 gagacacaag ctggctagcg tttaaacggg ccctctagac tcgagcacca tgaaaacgat
      781 catcgccctg agctacatct tctgcctggt attcgccatg tacaccgata tagagatgaa
      841 caggctggga aaggatagcc tcatgaacgg gaccgagggc ccaaacttct acgtgccttt
      901 ctccaacaag acgggcgtgg tgcgcagccc cttcgaggcc ccgcagtact acctggcgga
      961 gccatggcag ttctccatgc tggccgccta catgttcctg ctgatcatgc ttggcttccc
     1021 catcaacttc ctcacgctgt acgtcactgt gttccgtatg gcacgcacgg tgctcaacta
     1081 catcctgctc aacctggccg tggccgacct cttcatggtg ttcgggggct tcaccaccac
     1141 cctctacacc tctctgcacg ggtacttcgt ctttgggccc acgggctgca acctggaggg
     1201 cttctttgcc accttgggcg gtgaaattgc actgtggtcc ttggtggtcc tggccatcga
     1261 gcggtacgtg gtggtgctct accccgtctg ggccctgaac caccgcactg tgcagcgggc
     1321 catcatgggc gtcgccttca cctgggtcat ggctctggcc tgtgccgcgc cccccctcgt
     1381 cggctggtcc aggtacatcc cggagggcat gcagtgctcg tgcgggattg actactacac
     1441 gccccacgag gaaaccaaca atgagtcgtt cgtcatctac atgttcgtgg tccacttcat
     1501 catccccctg attgtcatat tcttctgcta cggactcatc cgggccaagc tcttgcggga
     1561 gggctgggtc catgccaacc ggcccaagcg catggtgatc atcatggtca tcgctttcct
     1621 aatctgctgg ctgccctacg ctggggtggc gttctacatc ttcacccatc agggctctga
     1681 ctttggcccc atcttcatga ccatcccggc tttctttgcc aagacttctg ccgtctacaa
     1741 ccccgtcatc tacatcatga tgaacaagca gttccgcgaa aagtttttcc agtctttgac
     1801 ttctgccctg gcgagggcgt ttggagagga ggagtttctg tcatcctgtc cccgtggcaa
     1861 cgccccccgg gaaacagaaa ccagccaagt ggcgcctgcc taagaattcc accacactgg
     1921 actagtggat ccgagctcgg taccaagctt aagtttaaac cgctgatcag cctcgactgt
     1981 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2041 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2101 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2161 agagaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2221 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2281 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2341 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2401 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2461 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2521 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2581 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2641 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     2701 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2761 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2821 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     2881 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     2941 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3001 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     3061 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3121 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3181 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3241 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     3301 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3361 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3421 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3481 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     3541 gtcgatcagg atgatctgga cgaagaacat caggggctcg cgccagccga actgttcgcc
     3601 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     3661 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     3721 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagaactt
     3781 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     3841 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     3901 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     3961 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4021 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4081 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4141 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4201 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4261 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4321 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4381 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4441 gctcttgcgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4501 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4561 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4621 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4681 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4741 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4801 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4861 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4921 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4981 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5041 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5101 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5161 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5221 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5281 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5341 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5401 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5461 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5521 cgcgggaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5581 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5641 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5701 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5761 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5821 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5881 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5941 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6001 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6061 cttcggggcg aaaactctca aggatcttac cactgttgag atccagttcg atgtaaccca
     6121 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6181 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6241 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6301 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6361 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     6421 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     6481 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     6541 c
//
