LOCUS       sequence-471052-        8061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-MBP-TEV-Rgs14 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8061)
  AUTHORS   Brown NE, Goswami D, Branch MR, Ramineni S, Ortlund EA, Griffin PR, 
            Hepler JR
  TITLE     Integration of G protein alpha (Galpha) signaling by the regulator 
            of G protein signaling 14 (RGS14).
  JOURNAL   J Biol Chem. 2015 Apr 3;290(14):9037-49. doi: 
            10.1074/jbc.M114.634329. Epub 2015 Feb 9.
  PUBMED    25666614
REFERENCE   2  (bases 1 to 8061)
  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: 
            "10.1074/jbc.M114.634329"; journalName: "J Biol Chem"; date: 
            "2015-04-3- 3"; volume: "290"; issue: "14"; pages: "9037-49"
COMMENT     Sequence Label: pLIC-His6-MBP-TEV-Rgs14
FEATURES             Location/Qualifiers
     source          1..8061
                     /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             5002..5019
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5023..6120
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      5977..5986
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     6094..6117
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             6178..6198
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             7905..7922
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7975..7993)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7989..8036
                     /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 tacatatgaa acaccatcac catcaccatg gcaaaatcga agaaggtaaa
     5041 ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa
     5101 ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa
     5161 ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
     5221 tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
     5281 gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac
     5341 ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
     5401 aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg
     5461 ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt
     5521 tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
     5581 ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
     5641 gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc
     5701 aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
     5761 ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt
     5821 aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact
     5881 gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
     5941 tacgaggaag agttggcgaa agatccacgt attgccgcca ccatggaaaa cgcccagaaa
     6001 ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg
     6061 gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact
     6121 aattcgagct cgaacaacaa caacaataac aataacaaca acagagatct gggtaccgag
     6181 aacctgtact tccaatccaa tgcgatgcca gggaagccca agcacttggg tgtccccaac
     6241 gggcgcatgg tcctggctgt ctctgatgga gaactgacca gcacatcagg ttcccaggcc
     6301 cagggtgagg gcagaggcag ctccctcagc atccacagcc tccccagtgg ccccagcagc
     6361 cccttctcca ctgatgagca gcctgtggcc agctgggccc agtcctttga gcggctgctg
     6421 caagacccac ggggtctggc ttacttcact gagttcctga agaaggaatt cagcgccgag
     6481 aatgtaactt tctggcaagc ctgcgagcgt ttccaacaga tcccagccag cgacaccaaa
     6541 cagctagctc aggaggccca caacatctac catgagttcc tgtccagcca ggcactgagc
     6601 ccagtgaaca tcgaccgaca ggcctggctt agtgaggagg tgctggccca gcccaggccc
     6661 gatatgttcc gagcacagca gcttcagatc ttcaatttga tgaagttcga cagctacgcg
     6721 cgcttcgtca aatccccgct gtaccaagag tgcctgctgg ccgaggccga gggacgcccc
     6781 ctgcgggaac ctggatcctc acacctcggg agcccggaca ctgcgaggaa gaagccaaag
     6841 ctgaagcctg gaaagtcact gccgctcggt gtggaagagt tggggcagct gccgcttgct
     6901 gagggccgcc ctctccgcaa gtcctttcgt agagagatgc cgggtggagc tgtgaattcg
     6961 gccctgcgac gagagtctca gggatccctg aattcttccg cgagtctgga cctgggcttc
     7021 cttgcctttg tgagcagcaa atccgagagc caccgaaaga gccttggaag tggagagggt
     7081 gagagcgaaa gccggcccgg gaagtactgc tgcgtgtatc tacctgatgg cacggcctcc
     7141 ttggccctgg ctcgacctgg cctcaccatc cgtgacatgc tggcaggcat ctgtgagaag
     7201 agaggcctct ctctacctga cattaaggtc tacctggtgg gcaaagaaca gaaggccctg
     7261 gtcctggatc aggactgcac cgtgctggca gaccaggaag tgcgactgga aaacaggatc
     7321 accttccagc tggagttggt cggcctggag cgtgtggtac ggatctctgc taagcccacc
     7381 aagcgtctgc aggaggcgct gcagcctatc ttggccaagc acggcctgag cctggatcag
     7441 gtggtcctac acaggccagg agagaagcag ctagtggatt tggagaatct agtgagctca
     7501 gtggcttcac agacactggt tttggacact cttccagatg caaagacaag ggaagccagc
     7561 agcatacctc cctgccgcag ccagggatgc ctccctagaa cccagaccaa ggacagtcac
     7621 cttcctccac tgtcctccag tttgtcggta gaagatgcca gtggttctac tgggaaacga
     7681 cagacctgcg acattgaagg cctagtggag ctgctgaatc gtgtgcagag cagcggggcc
     7741 catgaccaga ggggacttct tcgcaaagag gacctggtcc ttccagaatt tctgcagctt
     7801 ccttcccaaa gaccaggttc tcaggaggct ccaccatagc gcattggaag tggataacgg
     7861 atccgaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
     7921 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     7981 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     8041 aaggaggaac tatatccgga t
//
