LOCUS       sequence-457152-        4402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express human PHOSPHO2 protein (C-terminal Tobacco Etch Virus (TEV) 
            protease cleavable Twin-Strep-fusion 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 4402)
  TITLE     PHOSPHO2 is PA phosphatase and PHOSPHO1 is PLC
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4402)
  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            "Unpublished"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-PHOSPHO2-TEV-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..4402
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      133..721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     622..641
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     875..892
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        965..1042
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1043..2125
                     /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(1062..1081)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2174..2179,2180..2197,2198..2204)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2212..2228
                     /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     2217..2239
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             2979..2999
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(3009..3032,3033..3068,3069..3092)
                     /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(3146..3168)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3313..3331)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3399..3503
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3504..3572,3573..4364)
                     /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(3722..3741)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
       61 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
      121 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
      181 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      241 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
      301 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      361 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      421 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      481 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
      541 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
      601 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
      661 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
      721 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
      781 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
      841 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
      901 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataaa
      961 ttccgacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     1021 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     1081 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     1141 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     1201 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     1261 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     1321 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     1381 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     1441 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     1501 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     1561 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     1621 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     1681 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     1741 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     1801 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     1861 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     1921 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     1981 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccacgc tagcgcccaa
     2041 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     2101 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     2161 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     2221 gataacaatt tcacacagga aacagtatta tgaaaatttt gctagttttt gactttgaca
     2281 atacaatcat agatgacaat agtgacactt ggattgtaca atgtgctccc aacaaaaagc
     2341 ttcctattga actacgtgat tcttatcgaa aaggattttg gacagaattt atgggcagag
     2401 tctttaagta tttgggagat aagggtgtaa gagaacatga aatgaaaaga gcagtgacat
     2461 cattgccttt cactccaggg atggtggaac tcttcaactt tataagaaag aataaggata
     2521 aatttgactg cattattatt tcagattcaa attcggtctt catagattgg gttttagaag
     2581 ctgccagttt tcatgacata tttgataaag tgtttacaaa tccagcagct tttaatagca
     2641 atggtcatct cactgttgaa aattatcata ctcattcttg caatagatgc ccaaagaatc
     2701 tttgcaaaaa ggtagttttg atagaatttg tagataaaca gttacaacag ggagtgaatt
     2761 atacacaaat tgtttatatt ggtgatggtg gaaatgatgt ctgtccagtc acctttttaa
     2821 agaatgatga tgttgccatg ccacggaaag gatatacctt acagaaaact ctttccagaa
     2881 tgtctcaaaa tcttgagcct atggaatatt ctgttgtagt ttggtcctca ggtgttgata
     2941 taatttctca tttacaattt ctaataaagg atctcgagga aaacctctat tttcaaggcg
     3001 ccaccatgtg gtctcatccc cagttcgaaa aaggtggtgg aagtggtggg ggctcaggag
     3061 gatcagcctg gtcacaccct cagttcgaga agtgaagatc ttgactgacg atctgcctcg
     3121 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     3181 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     3241 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tataattctt
     3301 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3361 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3421 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3481 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3541 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3601 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3661 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3721 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     3781 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3841 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3901 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3961 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4021 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     4081 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4141 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4201 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4261 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4321 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4381 actcatatat actttagatt ga
//
