LOCUS       sequence-407823-        5910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express His tagged-RGS4 in bacterial cells for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5910)
  AUTHORS   Park JC, Luebbers A, Dao M, Semeano A, Nguyen AM, Papakonstantinou 
            MP, Broselid S, Yano H, Martemyanov KA, Garcia-Marcos M
  TITLE     Fine-tuning GPCR-mediated neuromodulation by biasing signaling 
            through different G protein subunits.
  JOURNAL   Mol Cell. 2023 Jul 20;83(14):2540-2558.e12. doi: 
            10.1016/j.molcel.2023.06.006. Epub 2023 Jun 29.
  PUBMED    37390816
REFERENCE   2  (bases 1 to 5910)
  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.molcel.2023.06.006"; journalName: "Mol Cell"; date: 
            "2023-07-20- 20"; volume: "83"; issue: "14"; pages: "2540-2558.e12"
COMMENT     Sequence Label: pLIC-His-RGS4
FEATURES             Location/Qualifiers
     source          1..5910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        285..388
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(389..457,458..1249)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(607..626)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1420..2008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1909..1928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2162..2179
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2194..2333
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2259..2278)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2419..2441
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2435..2626)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3435..4517)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4479..4498
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4518..4595)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4801..4820
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4908..4927
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4908..4926
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4927..4951
                     /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)."
     RBS             4966..4988
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4999..5016
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5041..5061
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5754..5771
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5824..5842)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5838..5885
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
      301 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
      361 tgcttcaata atttgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      421 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      481 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      541 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      601 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
      661 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      721 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      781 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      841 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      901 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      961 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1021 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1081 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1141 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1201 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1261 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1321 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1381 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1441 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1501 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1561 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1621 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1681 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1741 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1801 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1861 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1921 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1981 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2041 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2101 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2161 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2221 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     2281 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     2341 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2401 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2461 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     2521 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     2581 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     2641 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     2701 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     2761 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     2821 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     2881 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     2941 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     3001 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     3061 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     3121 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     3181 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     3241 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     3301 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3361 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     3421 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3481 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     3541 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     3601 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     3661 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     3721 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     3781 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     3841 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     3901 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     3961 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     4021 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     4081 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     4141 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     4201 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     4261 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     4321 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     4381 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     4441 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     4501 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     4561 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     4621 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     4681 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     4741 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     4801 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     4861 gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac
     4921 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     4981 gaaggagata tacatatgca ccatcatcat catcattctt ctggtgtaga tctgggtacc
     5041 gagaacctgt acttccaatc caatgcgatg tgcaaagggc ttgcaggtct gccggcttct
     5101 tgcttgagga gtgcaaaaga tatgaaacat cggctaggtt tcctgctgca aaaatctgat
     5161 tcctgtgaac acaattcttc ccacaacaag aaggacaaag tggttatttg ccagagagtg
     5221 agccaagagg aagtcaagaa atgggctgaa tcactggaaa acctgattag tcatgaatgt
     5281 gggctggcag ctttcaaagc tttcttgaag tctgaatata gtgaggagaa tattgacttc
     5341 tggatcagct gtgaagagta caagaaaatc aaatcaccat ctaaactaag tcccaaggcc
     5401 aaaaagatct ataatgaatt catctcagtc caggcaacca aagaggtgaa cctggattct
     5461 tgcaccaggg aagagacaag ccggaacatg ctagagccta caataacctg ctttgatgag
     5521 gcccagaaga agattttcaa cctgatggag aaggattcct accgccgctt cctcaagtct
     5581 cgattctatc ttgatttggt caacccgtcc agctgtgggg cagaaaagca gaaaggagcc
     5641 aagagttcag cagactgtgc ttccctggtc cctcagtgtg cctaatagcg cattggaagt
     5701 ggataacgga tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc
     5761 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     5821 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     5881 ttttgctgaa aggaggaact atatccggat
//
