LOCUS       sequence-127199-        5969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express His tagged GIV-CTs (1660–1870)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5969)
  AUTHORS   Garcia-Marcos M, Ear J, Farquhar MG, Ghosh P
  TITLE     A GDI (AGS3) and a GEF (GIV) regulate autophagy by balancing G 
            protein activity and growth factor signals.
  JOURNAL   Mol Biol Cell. 2011 Mar 1;22(5):673-86. doi: 
            10.1091/mbc.E10-08-0738. Epub 2011 Jan 5.
  PUBMED    21209316
REFERENCE   2  (bases 1 to 5969)
  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.E10-08-0738"; journalName: "Mol Biol Cell"; date: 
            "2011-03-1- 1"; volume: "22"; issue: "5"; pages: "673-86"
COMMENT     Sequence Label: His-GIV-CTs
FEATURES             Location/Qualifiers
     source          1..5969
                     /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(843..860)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(870..887)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             906..928
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    943..967
                     /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(967..986)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(968..986)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1070..1089)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1295..1372
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1373..2455
                     /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(1392..1411)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3264..3455
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3449..3471)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3557..3699
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3612..3631
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3714..3731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3885..4473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3965..3984)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4584..4603)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4595..5410
                     /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             4595..5410
                     /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(4668..4687)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5503..5958)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5640..5661)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5852..5871
                     /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 cgaattctta ttaggagctt tgttgctccc tagacctgct ttttgaattt
      241 ctgctttctt gtactttgtc cacattagat gctgcagtgg tgttgctgtc agcagcagct
      301 ggtggtgaat caactgatat aggcagtcta ctgtccttgg tcaaaaaatc atggatgctt
      361 gtccttcgtg tagttccttc ggcagttgag atcacgctgc ttgcacgagg taaagttgca
      421 taagggttac tatcttttga ttgtcgtgac agagaagatt cttttactaa ttttattttt
      481 ccttgagtgc ctggtgtagg ttttcccgca gaactaatga agtaggtatc ttcagttttt
      541 cgaggaccag gtctcaaaaa ctcaggctta gttgtccgtc tatcatagaa gtcccctggg
      601 gtttttccac ttactgacct ggccagacca cagctaactg gtttgtcttt tcccaaaaag
      661 tcagaagaga cagacaaact tttcattact tcatctaaaa gattctcttg acttgaggac
      721 tttatctgta ctgaggtaag cttattgctt tcttccaaaa actgttgtag agtaacaact
      781 tcacttccag gggaaccagt tttgatcttg tgatgtcgac tctccagtgt ttcagacata
      841 tggctgccgc gcggcaccag gccgctgctg tgatgatgat gatgatggct gctgcccatg
      901 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
      961 caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc
     1021 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
     1081 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
     1141 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
     1201 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
     1261 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
     1321 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     1381 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     1441 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     1501 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     1561 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     1621 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     1681 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     1741 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     1801 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     1861 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     1921 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     1981 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     2041 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     2101 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     2161 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     2221 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     2281 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     2341 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2401 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2461 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
     2521 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     2581 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
     2641 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
     2701 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     2761 gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc
     2821 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     2881 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     2941 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     3001 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     3061 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     3121 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     3181 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     3241 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     3301 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     3361 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     3421 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     3481 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     3541 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     3601 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa
     3661 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     3721 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3781 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3841 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3901 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3961 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4021 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4081 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4141 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4201 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4261 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4321 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4381 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4441 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4501 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact
     4561 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     4621 ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     4681 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc
     4741 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     4801 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     4861 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt
     4921 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     4981 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     5041 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     5101 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     5161 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     5221 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     5281 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     5341 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     5401 gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac
     5461 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgaaattgta aacgttaata
     5521 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5581 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5641 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5701 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5761 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5821 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5881 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5941 cgccgctaca gggcgcgtcc cattcgcca
//
