LOCUS       sequence-209703-        6988 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of a chimeric putative G-protein coupled receptor 
            (Rhodopsin-alpha1AR; Opto-alpha1AR; F6; 717).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6988)
  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 6988)
  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: "2018-05-16"; volume: "9(1):1950. doi"; pages: " 
            10.1038/s41467-018-04342-1"
COMMENT     Sequence Label: Opto-alpha1AR
FEATURES             Location/Qualifiers
     source          1..6988
                     /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(2418..2435)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2424..2648
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2694..3122
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2781..2800)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2991..3012
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3131..3151)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3136..3465
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3316..3451
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3378..3397
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3532..4326
                     /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(3586..3605)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4196..4215
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4500..4621
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4537..4556)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4591..4610
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4670..4686)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4670..4686)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4683..4705)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4694..4710
                     /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(4718..4724,4725..4742,4743..4748))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4763..4784
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4901..4918)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5072..5660)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5152..5171)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5831..6622,6623..6691))
                     /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     6454..6473
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6692..6796)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6871..6890)
                     /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 acgtcgtagc ctgtcaccga cacctgcact cagtcctcaa
     1081 ctacatcctg ctcaacctgg ccgtggccga cctcttcatg gtgttcgggg gcttcaccac
     1141 caccctctac acctctctgc acgggtactt cgtctttggg cccacgggct gcaacctgga
     1201 gggcttcttt gccaccttgg gcggtgaaat tgcactgtgg tccttggtgg tcctggccat
     1261 cgagcggtac gtggtggtga gctacccgct gcgctaccca accatcgtca cccagaggag
     1321 ggccatcatg ggcgtcgcct tcacctgggt catggctctg gcctgtgccg cgccccccct
     1381 cgtcggctgg tccaggtaca tcccggaggg catgcagtgc tcgtgcggga ttgactacta
     1441 cacgccccac gaggaaacca acaatgagtc gttcgtcatc tacatgttcg tggtccactt
     1501 catcatcccc ctgattgtca tattcttctg ctacggacgc gtctacgtgg tggccaagag
     1561 ggagagccgg ggcctcaagt ctggcctcaa gaccgacaag tcggactcgg agcaagtgac
     1621 gctccgcatc catcggaaaa acgccccggc aggaggcagc gggatggcca gcgccaagac
     1681 caagacgcac ttctcagtga ggctcctcaa gttctcccgg gagaagaaag cggcccgcat
     1741 ggtgatcatc atggtcatcg ctttcctaat ctgctggctg ccctacgctg gggtggcgtt
     1801 ctacatcttc acccatcagg gctctgactt tggccccatc ttcatgacca tcccggcttt
     1861 ctttgccaag acttctgccg tctacaaccc cgtcatctac atcatgatga acaagcagtt
     1921 ccgcaaggcc tttcagaatg tcttgagaat ccagtgtctc tgcagaaagc agtcctccaa
     1981 acatgccctg ggctacaccc tgcacccgcc cagccaggcc gtggaagggc aacacaagga
     2041 catggtgcgc atccccgtgg gatcaagaga aaccttctac aggatctcca agacggatgg
     2101 cgtttgtgaa tggaaatttt tctcttccat gccccgtgga tctgccagga ttacagtgtc
     2161 caaagaccaa tcctcctgta ccacagcccg ggtgagaagt aaaagctttt tgcaggtctg
     2221 ctgctgtgta gggccctcaa cccccagcct tgacaagaac catcaagttc caaccattaa
     2281 ggtccacacc atctccctca gtgagaacgg ggaggaagtc acagaaacca gccaagtggc
     2341 gcctgcctaa gaattccacc acactggact agtggatccg agctcggtac caagcttaag
     2401 tttaaaccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2461 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2521 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2581 gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg atgcggtggg
     2641 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2701 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2761 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2821 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2881 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2941 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3001 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3061 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3121 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3181 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3241 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3301 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3361 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3421 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3481 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     3541 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3601 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3661 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     3721 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3781 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3841 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3901 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3961 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agaacatcag
     4021 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4081 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4141 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4201 gctacccgtg atattgctga agaacttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4261 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4321 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4381 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4441 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4501 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4561 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4621 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4681 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4741 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4801 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4861 cggggagagg cggtttgcgt attgggcgct cttgcgcttc ctcgctcact gactcgctgc
     4921 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4981 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5041 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5101 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5161 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5221 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5281 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5341 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5401 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5461 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5521 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5581 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5641 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5701 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5761 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5821 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5881 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5941 catctggccc cagtgctgca atgataccgc gggacccacg ctcaccggct ccagatttat
     6001 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6061 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6121 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6181 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6241 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6301 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6361 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6421 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6481 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccac
     6541 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6601 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6661 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6721 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6781 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     6841 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     6901 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     6961 agctacaaca aggcaaggct tgaccgac
//
