LOCUS       sequence-457136-        3679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express C-terminal Tobacco Etch Virus (TEV) protease cleavable 
            Twin-Strep-tagged protein (ENLYFQGS-WSHPQFEK-(GGGS)2-GGSA-WSHPQFEK) 
            in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3679)
  TITLE     PHOSPHO2 is PA phosphatase and PHOSPHO1 is PLC
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3679)
  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            "Unpublished"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: pGEX-N-TEV-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..3679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(209..214,215..232,233..239)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    247..263
                     /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)."
     primer_bind     252..274
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             291..311
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(321..344,345..380,381..404)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     primer_bind     complement(458..480)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(625..643)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        711..815
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(816..884,885..1676)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1034..1053)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1847..2435
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2336..2355
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2589..2606
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2679..2756
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(2757..3679,1..160)
                     /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     complement(2776..2795)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac
       61 cacgctagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      121 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa
      181 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
      241 gtggaattgt gagcggataa caatttcaca caggaaacag tattctcgag gaaaacctct
      301 attttcaagg cgccaccatg tggtctcatc cccagttcga aaaaggtggt ggaagtggtg
      361 ggggctcagg aggatcagcc tggtcacacc ctcagttcga gaagtgaaga tcttgactga
      421 cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
      481 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
      541 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
      601 tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
      661 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
      721 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
      781 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
      841 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
      901 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
      961 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1021 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     1081 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1141 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1201 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1261 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1321 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     1381 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1441 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1501 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     1561 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     1621 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     1681 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     1741 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     1801 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     1861 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     1921 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     1981 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2041 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2101 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2161 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2221 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2281 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2341 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2401 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2461 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2521 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2581 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     2641 ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa
     2701 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     2761 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     2821 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     2881 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     2941 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     3001 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     3061 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     3121 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     3181 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     3241 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     3301 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     3361 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     3421 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     3481 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     3541 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     3601 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     3661 accgcttgct gcaactctc
//
