LOCUS       sequence-267725-        5463 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Streptococcus GB1 domain in vector for bacterial expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5463)
  AUTHORS   Zhou P, Lugovskoy AA, Wagner G
  TITLE     A solubility-enhancement tag (SET) for NMR studies of poorly 
            behaving proteins.
  JOURNAL   J Biomol NMR. 2001 May . 20(1):11-4.
  PUBMED    11430750
REFERENCE   2  (bases 1 to 5463)
  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: "J Biomol 
            NMR. 2001 May . 20(1):11-4."
COMMENT     Sequence Label: pET30-GBFusion1
FEATURES             Location/Qualifiers
     source          1..5463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        22..40
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    41..65
                     /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             80..102
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             116..277
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             284..301
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             345..362
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(415..433)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      429..476
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      513..968
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(600..619)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     810..831
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1060..1875)
                     /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             complement(1060..1875)
                     /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     1783..1802
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1867..1886
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1997..2585
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2486..2505
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2739..2756
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2771..2910
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2836..2855)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2996..3018
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3012..3203)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4012..5094)
                     /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     5056..5075
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5095..5172)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5378..5397
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
       61 attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata tgcagtacaa
      121 actgatcctg aacggtaaaa ccctgaaagg tgaaaccacc accgaagctg ttgacgctgc
      181 taccgcggaa aaagttttca aacagtacgc taacgacaac ggtgttgacg gtgaatggac
      241 ctacgacgac gctaccaaaa ccttcaccgt taccgaagga tcccaccacc accaccacca
      301 ctaagaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
      361 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
      421 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
      481 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
      541 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
      601 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
      661 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
      721 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
      781 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
      841 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
      901 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
      961 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1021 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     1081 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     1141 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     1201 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     1261 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     1321 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     1381 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     1441 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     1501 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     1561 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     1621 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     1681 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     1741 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     1801 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     1861 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     1921 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1981 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2041 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2101 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2161 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2221 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2281 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2341 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2401 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2461 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2521 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2581 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2641 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2701 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2761 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2821 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     2881 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     2941 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3001 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     3061 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     3121 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     3181 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     3241 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     3301 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     3361 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     3421 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     3481 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     3541 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     3601 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     3661 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     3721 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     3781 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     3841 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     3901 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     3961 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4021 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4081 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     4141 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     4201 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     4261 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     4321 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     4381 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     4441 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     4501 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     4561 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     4621 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     4681 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     4741 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     4801 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     4861 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     4921 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     4981 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     5041 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     5101 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     5161 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     5221 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     5281 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     5341 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     5401 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     5461 aga
//
