LOCUS       sequence-217211-        5759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and TF (Trigger factor (tig) from E. coli) fusions 
            with optional C-terminal StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5759)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5759)
  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            "unpublished"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-TF
FEATURES             Location/Qualifiers
     source          1..5759
                     /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             131..1423
                     /codon_start=1
                     /product="ribosome-associated chaperone from E. coli 
                     (Hoffmann et al., 2010)"
                     /label=Trigger Factor
                     /translation="QVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRK
                     GKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTY
                     SVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRV
                     TIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHA
                     ENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAI
                     RNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQA
                     KRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRN
                     VALEEQAVEAVLAKAKVTEKETTFNELMNQQA"
     CDS             1476..1499
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(1513..1531)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1514..1531)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1589..1607)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1603..1650
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1886..1907)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2057..3139)
                     /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     3101..3120
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3140..3217)
                     /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     3356..3375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3529..3548)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3648..3670
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3708..3726)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3794..3898
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3899..3967,3968..4759)
                     /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(4117..4136)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4930..5518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5419..5438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5672..5689
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc caagtttcag ttgaaaccac tcaaggcctt ggccgccgtg taacgattac
      181 tatcgctgct gacagcatcg aaaccgctgt taaaagcgag ctggtcaacg ttgcgaaaaa
      241 agtacgtatt gacggcttcc gcaaaggcaa agtgccaatg aatatcgttg ctcagcgtta
      301 tggcgcgtct gtacgccagg acgttctggg tgacctgatg agccgtaact tcattgacgc
      361 catcattaaa gaaaaaatca atccggctgg cgcaccgact tatgttccgg gcgaatacaa
      421 gctgggtgaa gacttcactt actctgtaga gtttgaagtt tatccggaag ttgaactgca
      481 gggtctggaa gcgatcgaag ttgaaaaacc gatcgttgaa gtgaccgacg ctgacgttga
      541 cggcatgctg gatactctgc gtaaacagca ggcgacctgg aaagaaaaag acggcgctgt
      601 tgaagcagaa gaccgcgtaa ccatcgactt caccggttct gtagacggcg aagagttcga
      661 aggcggtaaa gcgtctgatt tcgtactggc gatgggccag ggtcgtatga tcccgggctt
      721 tgaagacggt atcaaaggcc acaaagctgg cgaagagttc accatcgacg tgaccttccc
      781 ggaagaatac cacgcagaaa acctgaaagg taaagcagcg aaattcgcta tcaacctgaa
      841 gaaagttgaa gagcgtgaac tgccggaact gactgcagaa ttcatcaaac gtttcggcgt
      901 tgaagatggt tccgtagaag gtctgcgcgc tgaagtgcgt aaaaacatgg agcgcgagct
      961 gaagagcgcc atccgtaacc gcgttaagtc tcaggcgatc gaaggtctgg taaaagctaa
     1021 cgacatcgac gtaccggctg cgctgatcga cagcgaaatc gacgttctgc gtcgccaggc
     1081 tgcacagcgt ttcggtggca acgaaaaaca agctctggaa ctgccgcgcg aactgttcga
     1141 agaacaggct aaacgccgcg tagttgttgg cctgctgctg ggcgaagtta tccgcaccaa
     1201 cgagctgaaa gctgacgaag agcgcgtgaa aggcctgatc gaagagatgg cttctgcgta
     1261 cgaagatccg aaagaagtta tcgagttcta cagcaaaaac aaagaactga tggacaacat
     1321 gcgcaatgtt gctctggaag aacaggctgt tgaagctgta ctggcgaaag cgaaagtgac
     1381 tgaaaaagaa accactttca acgagctgat gaaccagcag gctagcggta ccgaaaacct
     1441 gtacttccag tgagacctta attaactcga gcgcatggag ccacccgcag ttcgaaaaat
     1501 aagcttgagt attctatagt gtcacctaaa tcccagcttg atccggctgc taacaaagcc
     1561 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     1621 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggataacct
     1681 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     1741 gcgaatggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     1801 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     1861 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     1921 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac agcttgcatg
     1981 cctgcaggtc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaacttac
     2041 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2101 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     2161 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     2221 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     2281 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     2341 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     2401 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     2461 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     2521 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     2581 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     2641 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     2701 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     2761 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     2821 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     2881 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     2941 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     3001 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     3061 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     3121 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     3181 ccataccgcg aaaggttttg caccattcga tggtgtcggc gagctcgaat tgtgaaccat
     3241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     3301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     3361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     3421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcctgatgc ggtattttct
     3481 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     3541 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     3601 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3661 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     3721 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     3781 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3841 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3901 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3961 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4021 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4081 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4141 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4201 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4261 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4321 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4381 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4441 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4501 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4561 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggttctcg
     4621 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4681 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4741 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4801 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4861 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4921 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4981 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5041 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5101 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5161 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5221 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5281 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5341 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5401 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5461 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5521 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5581 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5641 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     5701 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctgat
//
