LOCUS       sequence-252353-        4499 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 4499)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4499)
  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            "unpublished"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-Stag
FEATURES             Location/Qualifiers
     source          1..4499
                     /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             96..140
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             147..170
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             223..246
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(260..278)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(261..278)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(336..354)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      350..397
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(633..654)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(804..1886)
                     /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     1848..1867
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1887..1964)
                     /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     2103..2122
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2276..2295)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2395..2417
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2455..2473)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2541..2645
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2646..2714,2715..3506)
                     /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(2864..2883)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3677..4265
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4166..4185
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4419..4436
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agctgattaa tacgactcac tatagggaat tgtgagcgga taacaattcc tctagaaata
       61 attttgttta actttaagaa ggagatatat ccatgaaaga aaccgctgct gctaaattcg
      121 aacgccagca catggacagc actagtcacc atcaccatca ccatcaccat tctggtaccg
      181 aaaacctgta cttccagtga gaccttaatt aactcgagcg catggagcca cccgcagttc
      241 gaaaaataag cttgagtatt ctatagtgtc acctaaatcc cagcttgatc cggctgctaa
      301 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
      361 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
      421 ataacctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
      481 ctgaatggcg aatggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
      541 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
      601 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
      661 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccacagc
      721 ttgcatgcct gcaggtcgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
      781 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
      841 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
      901 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
      961 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     1021 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     1081 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     1141 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     1201 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     1261 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     1321 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     1381 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     1441 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     1501 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     1561 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     1621 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     1681 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     1741 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     1801 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     1861 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     1921 tatcatgcca taccgcgaaa ggttttgcac cattcgatgg tgtcggcgag ctcgaattgt
     1981 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2041 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2101 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2161 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ctgatgcggt
     2221 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     2281 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     2341 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     2401 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     2461 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     2521 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2581 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2641 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2701 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2761 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2821 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2881 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2941 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3001 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3061 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3121 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3181 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3241 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3301 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3361 gttctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3421 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3481 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3541 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3601 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3661 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3721 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3781 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     3841 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3901 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3961 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4021 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4081 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4141 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4201 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4261 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4321 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4381 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4441 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgc
//
