LOCUS       sequence-217230-        5312 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and Tsf (elongation factor Ts 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 5312)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5312)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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            "unpublished"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-Tsf
FEATURES             Location/Qualifiers
     source          1..5312
                     /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             1029..1052
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(1066..1084)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1067..1084)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1142..1160)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1156..1203
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1439..1460)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1610..2692)
                     /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     2654..2673
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2693..2770)
                     /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     2909..2928
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3082..3101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3201..3223
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3261..3279)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3347..3451
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3452..3520,3521..4312)
                     /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(3670..3689)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4483..5071
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4972..4991
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5225..5242
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc gcagaaatta cggcttccct ggtgaaagaa ttgcgcgaac gtacgggtgc
      181 aggcatgatg gactgcaaga aggcgctgac cgaagccaat ggcgacatcg agctggcgat
      241 cgagaacatg cgtaaatcgg gtgcaatcaa agcggccaaa aaggcgggta atgttgcggc
      301 agacggcgtc attaaaacca agatcgatgg taactatggc attattctgg aagtcaactg
      361 tcagacggat ttcgtggcga aggacgcggg ttttcaagcc ttcgccgaca aggtcctgga
      421 cgcagcggtg gctggtaaaa tcacggatgt cgaagtgttg aaagcacagt ttgaagaaga
      481 gcgtgtcgct ctggtggcaa agatcggcga gaatatcaat atccgccgtg ttgcggcact
      541 ggaaggtgat gttctgggca gctaccagca cggtgcgcgt attggcgttc tggtggctgc
      601 caagggtgcg gacgaagagc tggtgaaaca tatcgcgatg catgtcgccg cgagcaaacc
      661 agagtttatc aaaccggaag atgttagcgc cgaggtcgtg gaaaaagagt atcaggtaca
      721 gctggacatt gcgatgcagt ccggcaaacc taaagagatc gcagagaaga tggtcgaggg
      781 ccgcatgaag aagtttaccg gtgaggttag cctgactggt caaccgtttg ttatggagcc
      841 gagcaaaacg gttggtcaat tgctgaaaga gcacaatgcg gaagtgaccg gtttcattcg
      901 ttttgaggtg ggtgaaggca ttgagaaagt ggaaaccgat ttcgcggcag aagttgcggc
      961 tatgagcaag caatctagcg gtaccgaaaa cctgtacttc cagtgagacc ttaattaact
     1021 cgagcgcatg gagccacccg cagttcgaaa aataagcttg agtattctat agtgtcacct
     1081 aaatcccagc ttgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1141 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1201 ttgctgaaag gaggaactat atccggataa cctggcgtaa tagcgaagag gcccgcaccg
     1261 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa gcgttaatat
     1321 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     1381 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     1441 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     1501 cgtctatcag ggcgatggcc cacagcttgc atgcctgcag gtcggaagca taaagtgtaa
     1561 agcctggggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     1621 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     1681 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     1741 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     1801 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     1861 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     1921 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     1981 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     2041 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     2101 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     2161 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     2221 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     2281 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     2341 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     2401 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     2461 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     2521 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     2581 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     2641 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     2701 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt
     2761 cgatggtgtc ggcgagctcg aattgtgaac catcacccta atcaagtttt ttggggtcga
     2821 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2881 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2941 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     3001 cgctacaggg cgcgtcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3061 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3121 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3181 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3241 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3301 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3361 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3421 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3481 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3541 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3601 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3661 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3721 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3781 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3841 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3901 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3961 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4021 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4081 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4141 tgataaatct ggagccggtg agcgtggttc tcgcggtatc attgcagcac tggggccaga
     4201 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4261 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4321 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4381 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4441 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4501 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4561 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4621 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4681 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4741 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4801 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4861 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4921 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4981 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5041 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5101 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5161 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5221 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     5281 cgcgcgttgg ccgattcatt aatgcagctg at
//
