LOCUS       sequence-220671-        4438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as C-terminual 8His 
            fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4438)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4438)
  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            "unpublished"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-CHis
FEATURES             Location/Qualifiers
     source          1..4438
                     /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             155..178
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     promoter        complement(192..210)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(193..210)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(268..286)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      282..329
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(565..586)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(736..1818)
                     /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     1780..1799
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1819..1896)
                     /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     2035..2054
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2208..2227)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2327..2349
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2387..2405)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2473..2577
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2578..2646,2647..3438)
                     /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(2796..2815)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3609..4197
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4098..4117
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4351..4368
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgta tcgattaaat aaggaggaat aaaccatggg
      121 atccgaattc gcggccgccc taggctcgag ccctcatcac catcaccatc accatcacta
      181 agcttgagta ttctatagtg tcacctaaat cccagcttga tccggctgct aacaaagccc
      241 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
      301 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggataacctg
      361 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
      421 cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
      481 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
      541 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
      601 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccaca gcttgcatgc
      661 ctgcaggtcg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaacttaca
      721 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
      781 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
      841 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
      901 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
      961 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     1021 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     1081 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     1141 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     1201 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     1261 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     1321 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     1381 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     1441 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     1501 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     1561 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     1621 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     1681 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     1741 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     1801 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     1861 cataccgcga aaggttttgc accattcgat ggtgtcggcg agctcgaatt gtgaaccatc
     1921 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     1981 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     2041 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     2101 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcctgatgcg gtattttctc
     2161 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     2221 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     2281 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     2341 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     2401 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     2461 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2521 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2581 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2641 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     2701 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     2761 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     2821 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     2881 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     2941 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3001 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3061 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3121 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3181 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3241 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggttctcgc
     3301 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3361 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3421 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3481 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3541 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3601 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3661 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3721 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     3781 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3841 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3901 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3961 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4021 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4081 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4141 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4201 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4261 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4321 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4381 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctgat
//
