LOCUS       sequence-247819-        4916 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 4916)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4916)
  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            "unpublished"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-LSL
FEATURES             Location/Qualifiers
     source          1..4916
                     /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             633..656
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(670..688)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(671..688)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(746..764)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      760..807
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1043..1064)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1214..2296)
                     /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     2258..2277
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2297..2374)
                     /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     2513..2532
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2686..2705)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2805..2827
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2865..2883)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2951..3055
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3056..3124,3125..3916)
                     /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(3274..3293)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4087..4675
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4576..4595
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4829..4846
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc accgacatct acatcccgcc agaaggtctg tactttcgcc tcctgggttt
      181 cgcgtctcgt caggttatct tcgcgcgtaa ctctccttcc ccggacgttg gtctgagccc
      241 ggtgaatgat caggctaccg accagtattt ttctctgatc tatggtactg gtgaacatgc
      301 gggtctgtac gcgatcaaat ctaaagcgac cggcaaagtc ctgttctctc gtcgtccggc
      361 cgagccttat gtaggtcaga tcgatggtga tggtcgctac ccggacaatt ggttcaaaat
      421 cgaaccgggc aaaacttacc tgagcaaata cttccgcctg gtccagccgt ctacgggcac
      481 ggccctcgtt tctcgcaccc acctgcagcc gtatttctgg aaccacccgc agaccgaagt
      541 gttcgacgat caatatttca cctttctgtt cgaagacggt tccggtaccg aaaacctgta
      601 cttccagtga gaccttaatt aactcgagcg catggagcca cccgcagttc gaaaaataag
      661 cttgagtatt ctatagtgtc acctaaatcc cagcttgatc cggctgctaa caaagcccga
      721 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
      781 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg ataacctggc
      841 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
      901 aatggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
      961 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     1021 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     1081 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccacagc ttgcatgcct
     1141 gcaggtcgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aacttacatt
     1201 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     1261 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     1321 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     1381 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     1441 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     1501 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     1561 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     1621 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     1681 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     1741 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     1801 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     1861 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     1921 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     1981 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     2041 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     2101 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     2161 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     2221 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     2281 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     2341 taccgcgaaa ggttttgcac cattcgatgg tgtcggcgag ctcgaattgt gaaccatcac
     2401 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2461 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2521 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2581 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ctgatgcggt attttctcct
     2641 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     2701 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2761 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     2821 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     2881 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     2941 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3001 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3061 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3121 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3181 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3241 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3301 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3361 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3421 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3481 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3541 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3601 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3661 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3721 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gttctcgcgg
     3781 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3841 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3901 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3961 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4021 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4081 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4141 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4201 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4261 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4321 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4381 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4441 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4501 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4561 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4621 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4681 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4741 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4801 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4861 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctgat
//
