LOCUS       sequence-252870-        4892 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 4892)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4892)
  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            "unpublished"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-Spy
FEATURES             Location/Qualifiers
     source          1..4892
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..34
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        15..33
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    33..57
                     /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             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             106..129
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             623..646
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(660..678)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(661..678)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(736..754)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      750..797
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1033..1054)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1204..2286)
                     /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     2248..2267
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2287..2364)
                     /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     2503..2522
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2676..2695)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2795..2817
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2855..2873)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2941..3045
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3046..3114,3115..3906)
                     /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(3264..3283)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4077..4665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4566..4585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4819..4836
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ttaatgcagc tgattaatac gactcactat agggaattgt gagcggataa caattcctct
       61 agaaataatt ttgtttaact ttaagaagga gatatatcca tgaatcacca tcaccatcac
      121 catcaccatt ctggcactag tggtgcagac accactaccg cagcaccggc tgacgcgaag
      181 ccgatgatgc accacaaagg caagttcggt ccgcatcagg acatgatgtt caaagacctg
      241 aacctgaccg acgcgcagaa acagcagatc cgcgaaatca tgaaaggcca gcgtgaccag
      301 atgaaacgtc cgccgctgga agaacgccgc gcaatgcatg acatcattgc cagcgatacc
      361 ttcgataaag taaaagctga agcgcagatc gcaaaaatgg aagaacagcg caaagctaac
      421 atgctggcgc acatggaaac ccagaacaaa atttacaaca tcctgacgcc ggaacagaaa
      481 aagcaattta atgctaattt tgagaagcgt ctgacagaac gtccagcggc aaaaggtaaa
      541 atgcctgcaa ctgctgaaag tggtacctcc ggttctactg aaaacctgta cttccagtga
      601 gaccttaatt aactcgagcg catggagcca cccgcagttc gaaaaataag cttgagtatt
      661 ctatagtgtc acctaaatcc cagcttgatc cggctgctaa caaagcccga aaggaagctg
      721 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      781 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg ataacctggc gtaatagcga
      841 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggaaatt
      901 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
      961 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     1021 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     1081 aaagggcgaa aaaccgtcta tcagggcgat ggcccacagc ttgcatgcct gcaggtcgga
     1141 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aacttacatt aattgcgttg
     1201 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1261 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     1321 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     1381 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     1441 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     1501 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     1561 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     1621 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     1681 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     1741 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     1801 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     1861 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     1921 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     1981 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     2041 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     2101 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     2161 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     2221 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     2281 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     2341 ggttttgcac cattcgatgg tgtcggcgag ctcgaattgt gaaccatcac cctaatcaag
     2401 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     2461 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     2521 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     2581 cgcgcttaat gcgccgctac agggcgcgtc ctgatgcggt attttctcct tacgcatctg
     2641 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     2701 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2761 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2821 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     2881 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     2941 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3001 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3061 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3121 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3181 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3241 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     3301 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3361 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3421 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3481 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3541 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3601 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3661 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3721 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gttctcgcgg tatcattgca
     3781 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3841 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3901 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3961 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4021 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4081 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4141 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4201 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4261 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4321 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4381 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4441 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4501 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4561 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4621 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4681 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4741 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4801 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     4861 aaaccgcctc tccccgcgcg ttggccgatt ca
//
