LOCUS       sequence-252329-        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  JOURNAL   unpublished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            "unpublished"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     8..27
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8..26
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    26..50
                     /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             63..85
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             99..122
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             640..663
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(677..695)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(678..695)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(753..771)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      767..814
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1050..1071)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1221..2303)
                     /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     2265..2284
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2304..2381)
                     /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     2520..2539
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2693..2712)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2812..2834
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2872..2890)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2958..3062
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3063..3131,3132..3923)
                     /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(3281..3300)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4094..4682
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4583..4602
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4836..4853
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agctgattaa tacgactcac tatagggaat tgtgagcgga taacaattcc tctagaaata
       61 attttgttta actttaagaa ggagatatat ccatgaatca ccatcaccat caccatcacc
      121 attctggcac tagtggcacc gacatctaca tcccgccaga aggtctgtac tttcgcctcc
      181 tgggtttcgc gtctcgtcag gttatcttcg cgcgtaactc tccttccccg gacgttggtc
      241 tgagcccggt gaatgatcag gctaccgacc agtatttttc tctgatctat ggtactggtg
      301 aacatgcggg tctgtacgcg atcaaatcta aagcgaccgg caaagtcctg ttctctcgtc
      361 gtccggccga gccttatgta ggtcagatcg atggtgatgg tcgctacccg gacaattggt
      421 tcaaaatcga accgggcaaa acttacctga gcaaatactt ccgcctggtc cagccgtcta
      481 cgggcacggc cctcgtttct cgcacccacc tgcagccgta tttctggaac cacccgcaga
      541 ccgaagtgtt cgacgatcaa tatttcacct ttctgttcga agacggttcc ggtaccgaaa
      601 acctgtactt ccagtgagac cttaattaac tcgagcgcat ggagccaccc gcagttcgaa
      661 aaataagctt gagtattcta tagtgtcacc taaatcccag cttgatccgg ctgctaacaa
      721 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
      781 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata
      841 acctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
      901 aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
      961 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     1021 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     1081 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccacagcttg
     1141 catgcctgca ggtcggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     1201 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1261 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     1321 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     1381 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     1441 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     1501 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     1561 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     1621 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     1681 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     1741 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     1801 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     1861 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     1921 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     1981 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     2041 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     2101 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     2161 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     2221 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     2281 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     2341 catgccatac cgcgaaaggt tttgcaccat tcgatggtgt cggcgagctc gaattgtgaa
     2401 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     2461 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     2521 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     2581 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcctg atgcggtatt
     2641 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     2701 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     2761 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2821 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     2881 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     2941 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3001 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3061 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3121 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3181 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3241 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3301 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3361 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3421 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3481 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3541 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3601 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3661 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3721 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggtt
     3781 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3841 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3901 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3961 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4021 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4081 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4141 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4201 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4261 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4321 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4381 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4441 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4501 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4561 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4621 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4681 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4741 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4801 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4861 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgc
//
