LOCUS       sequence-247815-        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  JOURNAL   unpublished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            "unpublished"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"
     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             441..464
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(478..496)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(479..496)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(554..572)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      568..615
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(851..872)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1022..2104)
                     /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     2066..2085
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2105..2182)
                     /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     2321..2340
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2494..2513)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2613..2635
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2673..2691)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2759..2863
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2864..2932,2933..3724)
                     /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(3082..3101)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3895..4483
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4384..4403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4637..4654
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggt gctttcgaat tcaaactgcc ggacatcggt gaaggtatcc acgaaggtga
      181 aatcgttaaa tggtttgtta aaccgggtga cgaagttaac gaagacgacg ttctgtgcga
      241 agttcagaac gacaaagcgg ttgttgaaat cccgtctccg gttaaaggta aagttctgga
      301 aatcctggtt ccggagggta ctgttgcgac cgttggtcag accctgatca ccctggacgc
      361 gccaggttac gaaaacatga ctaccggcag cggtaccgaa aacctgtact tccagtgaga
      421 ccttaattaa ctcgagcgca tggagccacc cgcagttcga aaaataagct tgagtattct
      481 atagtgtcac ctaaatccca gcttgatccg gctgctaaca aagcccgaaa ggaagctgag
      541 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      601 ttgaggggtt ttttgctgaa aggaggaact atatccggat aacctggcgt aatagcgaag
      661 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt
      721 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
      781 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
      841 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
      901 agggcgaaaa accgtctatc agggcgatgg cccacagctt gcatgcctgc aggtcggaag
      961 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     1021 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1081 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     1141 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     1201 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     1261 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     1321 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     1381 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     1441 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     1501 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     1561 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     1621 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     1681 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     1741 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     1801 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     1861 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     1921 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     1981 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     2041 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     2101 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     2161 ttttgcacca ttcgatggtg tcggcgagct cgaattgtga accatcaccc taatcaagtt
     2221 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2281 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2341 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2401 cgcttaatgc gccgctacag ggcgcgtcct gatgcggtat tttctcctta cgcatctgtg
     2461 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     2521 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2581 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     2641 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     2701 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     2761 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2821 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2881 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2941 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     3001 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     3061 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     3121 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     3181 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3241 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3301 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3361 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3421 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3481 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3541 ctggtttatt gctgataaat ctggagccgg tgagcgtggt tctcgcggta tcattgcagc
     3601 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3661 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3721 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3781 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3841 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3901 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3961 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4021 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4081 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4141 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4201 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4261 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4321 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4381 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4441 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4501 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4561 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4621 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     4681 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tgat
//
