LOCUS       sequence-217228-        4631 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and GB1 fusions with optional C-terminal 
            StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4631)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4631)
  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"; journalName: 
            "unpublished"
COMMENT     Sequence Label: pExp-GB1
FEATURES             Location/Qualifiers
     source          1..4631
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..115
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             134..295
                     /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             348..371
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(385..403)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(386..403)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(461..479)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      475..522
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(758..779)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(929..2011)
                     /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     1973..1992
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2012..2089)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     2228..2247
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2401..2420)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2520..2542
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2580..2598)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2666..2770
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2771..2839,2840..3631)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2989..3008)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3802..4390
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4291..4310
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4544..4561
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc acctacaaac tgatcctgaa cggtaaaacc ctgaaaggtg aaaccaccac
      181 cgaagctgta gacgctgcta ctgctgaaaa agttttcaaa cagtacgcta acgacaacgg
      241 tgtggacggt gaatggacct acgacgacgc taccaaaacc ttcacggtta cggaaagcgg
      301 taccgaaaac ctgtacttcc agtgagacct taattaactc gagcgcatgg agccacccgc
      361 agttcgaaaa ataagcttga gtattctata gtgtcaccta aatcccagct tgatccggct
      421 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
      481 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      541 tccggataac ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
      601 gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
      661 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
      721 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
      781 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
      841 acagcttgca tgcctgcagg tcggaagcat aaagtgtaaa gcctggggtg cctaatgagt
      901 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
      961 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1021 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     1081 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     1141 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     1201 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     1261 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     1321 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     1381 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     1441 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     1501 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     1561 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     1621 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     1681 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     1741 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     1801 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     1861 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     1921 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     1981 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     2041 ggcgctatca tgccataccg cgaaaggttt tgcaccattc gatggtgtcg gcgagctcga
     2101 attgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     2161 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2221 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2281 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcctgat
     2341 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     2401 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     2461 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2521 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     2581 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     2641 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2701 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2761 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2821 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2881 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2941 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3001 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3061 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3121 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3181 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3241 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3301 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3361 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3421 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3481 gcgtggttct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3541 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3601 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3661 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3721 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3781 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3841 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3901 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3961 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4021 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4081 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4141 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4201 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4261 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4321 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4381 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4441 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4501 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4561 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     4621 atgcagctga t
//
