LOCUS       sequence-252354-        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  JOURNAL   unpublished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            "unpublished"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     10..29
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        10..28
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    28..52
                     /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             65..87
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             95..127
                     /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             134..157
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             210..233
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(247..265)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(248..265)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(323..341)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      337..384
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(620..641)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(791..1873)
                     /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     1835..1854
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1874..1951)
                     /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     2090..2109
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2263..2282)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2382..2404
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2442..2460)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2528..2632
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2633..2701,2702..3493)
                     /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(2851..2870)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3664..4252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4153..4172
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4406..4423
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcagctgatt aatacgactc actataggga attgtgagcg gataacaatt cctctagaaa
       61 taattttgtt taactttaag aaggagatat atccatggct agcatgactg gtggacagca
      121 aatgggtact agtcaccatc accatcacca tcaccattct ggtaccgaaa acctgtactt
      181 ccagtgagac cttaattaac tcgagcgcat ggagccaccc gcagttcgaa aaataagctt
      241 gagtattcta tagtgtcacc taaatcccag cttgatccgg ctgctaacaa agcccgaaag
      301 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
      361 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata acctggcgta
      421 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
      481 ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
      541 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      601 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      661 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccacagcttg catgcctgca
      721 ggtcggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac ttacattaat
      781 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
      841 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
      901 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
      961 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     1021 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     1081 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     1141 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     1201 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     1261 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     1321 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     1381 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     1441 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     1501 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     1561 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     1621 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     1681 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     1741 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     1801 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     1861 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     1921 cgcgaaaggt tttgcaccat tcgatggtgt cggcgagctc gaattgtgaa ccatcaccct
     1981 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     2041 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     2101 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     2161 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcctg atgcggtatt ttctccttac
     2221 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     2281 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     2341 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     2401 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     2461 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     2521 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2581 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     2641 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2701 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2761 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2821 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     2881 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2941 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3001 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3061 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3121 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3181 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3241 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     3301 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggtt ctcgcggtat
     3361 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     3421 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3481 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3541 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3601 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3661 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3721 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3781 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     3841 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3901 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3961 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4021 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4081 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4141 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4201 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4261 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4321 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4381 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     4441 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taat
//
