LOCUS       sequence-247828-        4472 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4472)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4472)
  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            "unpublished"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-Etag
FEATURES             Location/Qualifiers
     source          1..4472
                     /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             113..136
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             189..212
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(226..244)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(227..244)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(302..320)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      316..363
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(599..620)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(770..1852)
                     /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     1814..1833
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1853..1930)
                     /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     2069..2088
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2242..2261)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2361..2383
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2421..2439)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2507..2611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2612..2680,2681..3472)
                     /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(2830..2849)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3643..4231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4132..4151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4385..4402
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgac agatgtaacg attaaaacta gtcaccatca
      121 ccatcaccat caccattctg gtaccgaaaa cctgtacttc cagtgagacc ttaattaact
      181 cgagcgcatg gagccacccg cagttcgaaa aataagcttg agtattctat agtgtcacct
      241 aaatcccagc ttgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
      301 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      361 ttgctgaaag gaggaactat atccggataa cctggcgtaa tagcgaagag gcccgcaccg
      421 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa gcgttaatat
      481 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
      541 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
      601 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
      661 cgtctatcag ggcgatggcc cacagcttgc atgcctgcag gtcggaagca taaagtgtaa
      721 agcctggggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
      781 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
      841 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
      901 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
      961 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     1021 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     1081 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     1141 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     1201 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     1261 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     1321 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     1381 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     1441 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     1501 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     1561 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     1621 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     1681 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     1741 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     1801 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     1861 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt
     1921 cgatggtgtc ggcgagctcg aattgtgaac catcacccta atcaagtttt ttggggtcga
     1981 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2041 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2101 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2161 cgctacaggg cgcgtcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2221 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     2281 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2341 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2401 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     2461 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2521 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2581 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     2641 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     2701 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     2761 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     2821 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     2881 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     2941 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3001 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3061 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3121 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3181 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3241 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3301 tgataaatct ggagccggtg agcgtggttc tcgcggtatc attgcagcac tggggccaga
     3361 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3421 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3481 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3541 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     3601 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3661 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3721 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3781 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3841 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3901 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3961 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4021 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4081 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4141 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4201 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4261 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4321 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4381 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     4441 cgcgcgttgg ccgattcatt aatgcagctg at
//
