LOCUS       sequence-220196-        4822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as Thrombin cleavable 
            N-terminal 6His-tag-thioredoxin fusions .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4822)
  AUTHORS   Peranen J, Rikkonen M, Hyvonen M, Kaariainen L
  TITLE     T7 vectors with modified T7lac promoter for expression of proteins 
            in Escherichia coli.
  JOURNAL   Anal Biochem. 1996 May 1;236(2):371-3.
  PUBMED    8660525
REFERENCE   2  (bases 1 to 4822)
  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: "Anal 
            Biochem."; date: "1996-05-1"; volume: "236(2)"; pages: "371-3"
COMMENT     Sequence Label: pTAT5
FEATURES             Location/Qualifiers
     source          1..4822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..45
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     116..135
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        116..134
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    134..158
                     /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             171..193
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             213..230
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             240..566
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             597..614
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     promoter        complement(687..705)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(688..705)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(763..781)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      777..824
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1060..1081)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1231..2313)
                     /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     2275..2294
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2314..2391)
                     /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     2534..2553
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2707..2726)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2826..2848
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2886..2904)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2972..3076
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3077..3145,3146..3937)
                     /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(3295..3314)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4108..4696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4597..4616
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
       61 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctgattaata
      121 cgactcacta tagggaattg tgagcggata acaattcctc tagaaataat tttgtttaac
      181 tttaagaagg agatatatcc atgaacacca ttcatcacca tcaccatcac aacactagta
      241 tgagcgataa aattattcac ctgactgacg acagttttga cacggatgta ctcaaagcgg
      301 acggggcgat cctcgtcgat ttctgggcag agtggtgcgg tccgtgcaaa atgatcgccc
      361 cgattctgga tgaaatcgct gacgaatatc agggcaaact gaccgttgca aaactgaaca
      421 tcgatcaaaa ccctggcact gcgccgaaat atggcatccg tggtatcccg actctgctgc
      481 tgttcaaaaa cggtgaagtg gcggcaacca aagtgggtgc actgtctaaa ggtcagttga
      541 aagagttcct cgacgctaac ctggcgacta gtggatctgg tggtggtggc ggccgcctgg
      601 ttccgagggg atccatggat atcgaattcg tcgacctcga gggatccggg ccctctagat
      661 gcggccgcat gcataagctt gagtattcta tagtgtcacc taaatcccag cttgatccgg
      721 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
      781 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
      841 tatccggata acctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
      901 gcgcagcctg aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta
      961 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     1021 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     1081 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     1141 ccacagcttg catgcctgca ggtcggaagc ataaagtgta aagcctgggg tgcctaatga
     1201 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1261 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1321 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     1381 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     1441 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     1501 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     1561 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     1621 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     1681 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     1741 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     1801 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     1861 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     1921 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     1981 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     2041 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     2101 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     2161 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     2221 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     2281 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     2341 cgggcgctat catgccatac cgcgaaaggt tttgcaccat tcgatggtgt cgggtaccga
     2401 gctcgaattg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     2461 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     2521 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     2581 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     2641 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     2701 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     2761 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     2821 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     2881 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     2941 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3001 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3061 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3121 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3181 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3241 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3301 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3361 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3421 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3481 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3541 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     3601 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     3661 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     3721 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     3781 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3841 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3901 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     3961 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4021 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4081 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4141 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4201 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4261 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4321 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4381 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4441 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4501 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4561 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4621 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4681 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4741 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4801 cgcctttgag tgagctgata cc
//
