LOCUS       sequence-252322-        4724 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 4724)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4724)
  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            "unpublished"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-Lipo
FEATURES             Location/Qualifiers
     source          1..4724
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             38..60
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             74..97
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             423..446
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(460..478)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(461..478)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(536..554)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      550..597
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(833..854)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1004..2086)
                     /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     2048..2067
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2087..2164)
                     /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     2303..2322
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2476..2495)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2595..2617
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2655..2673)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2741..2845
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2846..2914,2915..3706)
                     /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(3064..3083)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3877..4465
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4366..4385
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4619..4636
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(4707..4724,1..2)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4707..4724,1)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcctctag aaataatttt gtttaacttt aagaaggaga
       61 tatatccatg aatcaccatc accatcacca tcaccattct ggcactagtg gtgctttcga
      121 attcaaactg ccggacatcg gtgaaggtat ccacgaaggt gaaatcgtta aatggtttgt
      181 taaaccgggt gacgaagtta acgaagacga cgttctgtgc gaagttcaga acgacaaagc
      241 ggttgttgaa atcccgtctc cggttaaagg taaagttctg gaaatcctgg ttccggaggg
      301 tactgttgcg accgttggtc agaccctgat caccctggac gcgccaggtt acgaaaacat
      361 gactaccggc agcggtaccg aaaacctgta cttccagtga gaccttaatt aactcgagcg
      421 catggagcca cccgcagttc gaaaaataag cttgagtatt ctatagtgtc acctaaatcc
      481 cagcttgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      541 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      601 aaaggaggaa ctatatccgg ataacctggc gtaatagcga agaggcccgc accgatcgcc
      661 cttcccaaca gttgcgcagc ctgaatggcg aatggaaatt gtaagcgtta atattttgtt
      721 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
      781 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
      841 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
      901 tcagggcgat ggcccacagc ttgcatgcct gcaggtcgga agcataaagt gtaaagcctg
      961 gggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     1021 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1081 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     1141 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     1201 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     1261 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     1321 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     1381 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     1441 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     1501 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     1561 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     1621 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     1681 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     1741 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     1801 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     1861 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     1921 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     1981 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     2041 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     2101 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac cattcgatgg
     2161 tgtcggcgag ctcgaattgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     2221 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     2281 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     2341 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     2401 agggcgcgtc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     2461 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     2521 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     2581 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     2641 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     2701 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2761 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2821 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2881 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2941 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3001 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3061 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     3121 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3181 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3241 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3301 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3361 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     3421 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3481 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3541 atctggagcc ggtgagcgtg gttctcgcgg tatcattgca gcactggggc cagatggtaa
     3601 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3661 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3721 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3781 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3841 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3901 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3961 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4021 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4081 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4141 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4201 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4261 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4321 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4381 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4441 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4501 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4561 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4621 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     4681 ttggccgatt cattaatgca gctgattaat acgactcact atag
//
