LOCUS       sequence-148165-        6398 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His tagged GINA3 protein in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6398)
  AUTHORS   Lin C, Ear J, Midde K, Lopez-Sanchez I, Aznar N, Garcia-Marcos M, 
            Kufareva I, Abagyan R, Ghosh P
  TITLE     Structural basis for activation of trimeric Gi proteins by multiple 
            growth factor receptors via GIV/Girdin.
  JOURNAL   Mol Biol Cell. 2014 Nov 5;25(22):3654-71. doi: 
            10.1091/mbc.E14-05-0978. Epub 2014 Sep 3.
  PUBMED    25187647
REFERENCE   2  (bases 1 to 6398)
  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: 
            "10.1091/mbc.E14-05-0978"; journalName: "Mol Biol Cell"; date: 
            "2014-11-5- 5"; volume: "25"; issue: "22"; pages: "3654-71"
COMMENT     Sequence Label: His-GINA3
FEATURES             Location/Qualifiers
     source          1..6398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1272..1289)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1299..1316)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1335..1357
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1372..1396
                     /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)."
     primer_bind     complement(1396..1415)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1397..1415)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1499..1518)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1724..1801
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1802..2884
                     /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     complement(1821..1840)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3693..3884
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3878..3900)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3986..4128
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4041..4060
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4143..4160)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4314..4902)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4394..4413)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5013..5032)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5024..5839
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5024..5839
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5097..5116)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5932..6387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6069..6090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6281..6300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tcctcagtaa agcccacatt cctttaagtt gtttttaatg
      241 atgacatctg taacagcatc aaaaacaaac tgcacgtttt tggtgtcagt ggcacaggta
      301 aagtgagtgt agacctcctt ggtgtccttt cttcggttca gatcttcaaa ctggcactgg
      361 atgtaagcag ctgcctcttc gtacgtattg gaacctgtgt attctggata acagattgtt
      421 aatggactcc tctttatttt ttcctcaaaa aggtctttct tattaaggaa gagaatgatt
      481 gaagtgtctg taaaccattt gttgttacaa atgctgtcaa acaatttcat gctttcatgc
      541 attcggttca tttcctcgtc ctcagccaga acaaggtcgt aatcactgag agccacacaa
      601 aagataattg ctgtcactcc ctcaaaacag tggatccact ttttccgttc ggatctttgg
      661 ccacctacat caaacatttt gaagtagagc tccttgaagg tgaagtgggt ctccacaatg
      721 cctgtagtct tcactctcgt ccgaagaaca tcttgctgag ttggaatgta gttggtctgg
      781 gatattctat ccaaatcatt taggtaatat gaagcagaat cattgagctg atattccctg
      841 gacctgctga agcatgcctg caccccgcca tctcgccata aacgtttaat cacgcctgct
      901 agttctgaag tcatgactcc ttcctcagca ctgccagcta aaacaaataa ctgtcgggca
      961 tcatccgctc tggcagcttc cccaaaatca atcttcaacc gtcccatggc tcttatgatt
     1021 gcaatgatgg actgaatggt attgctgtag acgacaactt tatactgctt acactcgtcc
     1081 tcggaatagc catcctcatg aatgattttc atctgtttca caatagtact tttaccagat
     1141 tctccagcgc cgagcagcag cagcttcact tctttggccg ctttctctcc gtcctcccgc
     1201 aagttgcggt cgatcatctt actccgctcc accgccgcct tgtcctcggc gctcaacgtg
     1261 cagcccatat ggctgccgcg cggcaccagg ccgctgctgt gatgatgatg atgatggctg
     1321 ctgcccatgg tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt
     1381 atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc
     1441 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
     1501 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
     1561 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
     1621 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
     1681 ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg
     1741 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     1801 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     1861 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     1921 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     1981 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     2041 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     2101 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     2161 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     2221 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     2281 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     2341 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     2401 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     2461 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     2521 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     2581 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     2641 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     2701 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     2761 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     2821 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2881 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga
     2941 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     3001 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     3061 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     3121 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     3181 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga
     3241 tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga
     3301 atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct
     3361 gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt
     3421 cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg
     3481 gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt
     3541 cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt
     3601 catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
     3661 tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta
     3721 acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg
     3781 acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc
     3841 gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     3901 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     3961 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     4021 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatatg
     4081 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     4141 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4201 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4261 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4321 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4381 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4441 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4501 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4561 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4621 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4681 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4741 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4801 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4861 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4921 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac
     4981 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
     5041 ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     5101 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa
     5161 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     5221 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     5281 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc
     5341 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     5401 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     5461 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     5521 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt
     5581 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     5641 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     5701 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     5761 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     5821 gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga
     5881 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaaattgtaa
     5941 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     6001 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     6061 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     6121 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     6181 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     6241 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     6301 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     6361 cgcttaatgc gccgctacag ggcgcgtccc attcgcca
//
