LOCUS       sequence-247826-        4484 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 4484)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4484)
  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            "unpublished"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-T7tag
FEATURES             Location/Qualifiers
     source          1..4484
                     /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             86..118
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             125..148
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             201..224
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(238..256)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(239..256)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(314..332)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      328..375
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(611..632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(782..1864)
                     /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     1826..1845
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1865..1942)
                     /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     2081..2100
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2254..2273)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2373..2395
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2433..2451)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2519..2623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2624..2692,2693..3484)
                     /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(2842..2861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3655..4243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4144..4163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4397..4414
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatggc tagcatgact ggtggacagc aaatgggtac
      121 tagtcaccat caccatcacc atcaccattc tggtaccgaa aacctgtact tccagtgaga
      181 ccttaattaa ctcgagcgca tggagccacc cgcagttcga aaaataagct tgagtattct
      241 atagtgtcac ctaaatccca gcttgatccg gctgctaaca aagcccgaaa ggaagctgag
      301 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      361 ttgaggggtt ttttgctgaa aggaggaact atatccggat aacctggcgt aatagcgaag
      421 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt
      481 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
      541 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
      601 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
      661 agggcgaaaa accgtctatc agggcgatgg cccacagctt gcatgcctgc aggtcggaag
      721 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
      781 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
      841 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
      901 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
      961 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     1021 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     1081 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     1141 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     1201 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     1261 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     1321 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     1381 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     1441 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     1501 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     1561 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     1621 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     1681 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     1741 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     1801 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     1861 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     1921 ttttgcacca ttcgatggtg tcggcgagct cgaattgtga accatcaccc taatcaagtt
     1981 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2041 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2101 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2161 cgcttaatgc gccgctacag ggcgcgtcct gatgcggtat tttctcctta cgcatctgtg
     2221 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     2281 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2341 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     2401 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     2461 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     2521 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2581 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2641 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2701 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2761 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2821 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2881 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2941 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3001 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3061 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3121 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3181 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3241 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3301 ctggtttatt gctgataaat ctggagccgg tgagcgtggt tctcgcggta tcattgcagc
     3361 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3421 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3481 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3541 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3601 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3661 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3721 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3781 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3841 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3901 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3961 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4021 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4081 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4141 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4201 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4261 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4321 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4381 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     4441 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tgat
//
