LOCUS       sequence-217235-        4481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His fusions with optional C-terminal StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4481)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4481)
  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            "unpublished"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-NHis
FEATURES             Location/Qualifiers
     source          1..4481
                     /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             86..112
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             122..145
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             198..221
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(235..253)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(236..253)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(311..329)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      325..372
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(608..629)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(779..1861)
                     /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     1823..1842
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1862..1939)
                     /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     2078..2097
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2251..2270)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2370..2392
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2430..2448)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2516..2620
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2621..2689,2690..3481)
                     /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(2839..2858)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3652..4240
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4141..4160
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4394..4411
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgta tcgattaaat aaggaggaat aaaccatgaa
      121 tcaccatcac catcaccatc accattctgg taccgaaaac ctgtacttcc agtgagacct
      181 taattaactc gagcgcatgg agccacccgc agttcgaaaa ataagcttga gtattctata
      241 gtgtcaccta aatcccagct tgatccggct gctaacaaag cccgaaagga agctgagttg
      301 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      361 aggggttttt tgctgaaagg aggaactata tccggataac ctggcgtaat agcgaagagg
      421 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag
      481 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
      541 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
      601 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
      661 gcgaaaaacc gtctatcagg gcgatggccc acagcttgca tgcctgcagg tcggaagcat
      721 aaagtgtaaa gcctggggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
      781 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      841 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
      901 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
      961 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     1021 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     1081 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     1141 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     1201 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     1261 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     1321 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     1381 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     1441 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     1501 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     1561 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     1621 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     1681 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     1741 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     1801 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     1861 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     1921 tgcaccattc gatggtgtcg gcgagctcga attgtgaacc atcaccctaa tcaagttttt
     1981 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     2041 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     2101 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     2161 ttaatgcgcc gctacagggc gcgtcctgat gcggtatttt ctccttacgc atctgtgcgg
     2221 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2281 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2341 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     2401 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     2461 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     2521 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2581 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2641 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2701 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     2761 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     2821 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     2881 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     2941 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3001 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3061 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3121 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3181 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3241 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3301 gtttattgct gataaatctg gagccggtga gcgtggttct cgcggtatca ttgcagcact
     3361 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3421 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     3481 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3541 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3601 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3661 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3721 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3781 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3841 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3901 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3961 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4021 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4081 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4141 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4201 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4261 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4321 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4381 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     4441 gcctctcccc gcgcgttggc cgattcatta atgcagctga t
//
