LOCUS       sequence-322059-        7034 bp    DNA     circular SYN 14-APR-2025
DEFINITION  bacterial expression of MBP-TEV n-terminal fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7034)
  AUTHORS   Currinn H, Guscott B, Balklava Z, Rothnie A, Wassmer T
  TITLE     APP controls the formation of PI(3,5)P vesicles through its binding 
            of the PIKfyve complex.
  JOURNAL   Cell Mol Life Sci. 2015 Jul 28.
  PUBMED    26216398
REFERENCE   2  (bases 1 to 7034)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Mol 
            Life Sci. 2015 Jul 28."
COMMENT     Sequence Label: pET28-MBP-TEV
FEATURES             Location/Qualifiers
     source          1..7034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             335..352
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             368..1465
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1322..1331
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1439..1462
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1481..1501
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2107..2124
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2177..2195)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2191..2238
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2275..2730
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2362..2381)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2572..2593
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2822..3637)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2822..3637)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3545..3564
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3629..3648
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3759..4347
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4248..4267
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4501..4518
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4533..4672
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4598..4617)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4758..4780
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4774..4965)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5774..6856)
                     /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     6818..6837
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6857..6934)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatggc
      361 tagcggtaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
      421 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
      481 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
      541 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
      601 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
      661 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
      721 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
      781 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
      841 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
      901 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
      961 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
     1021 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
     1081 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
     1141 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcgaaaga
     1201 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
     1261 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc
     1321 cgccaccatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
     1381 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga
     1441 tgaagccctg aaagacgcgc agactcgtat caccaagggt gaaaatctat acttccaagg
     1501 atccctcagt acaggctgct actgacagat tacttaaaga accttctaga agactctgta
     1561 gatcacagag acacccaaga tgccctggct gttgtcatag aggtagctaa ccatgccaat
     1621 gacaccatga agcaagggga caactttcag aagctcatgc agattcagta cagcttaagt
     1681 ggacaccatg aaattgtgca acctgggcgg gtttttctta aagaaggcac cctgatgaag
     1741 ctgtctcgga aagtcatgca gccccggatg tttttcctgt ttaacgatgc tcttctctac
     1801 accacaccca tgcagtctgg gatgtacaaa ctgaacaaca tgctctcgct ggctggaatg
     1861 aaggtcagaa aacctaccca ggaggcatat cagaacgagt taaagattga aagtgtagag
     1921 cgttctttca tcctctcagc tagttctgcc gcagaaagag acgactggct ggaggccata
     1981 tccagctcca tagaagagta tgccaagaaa agaatcacat tctgccccag caggagtctt
     2041 gatgaggact cagaaaggaa agaagaagtt agccccctcg gggcaaaggc tccctaaccg
     2101 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     2161 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     2221 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     2281 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2341 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2401 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2461 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2521 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2581 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2641 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2701 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     2761 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     2821 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     2881 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     2941 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     3001 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     3061 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     3121 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     3181 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     3241 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     3301 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     3361 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     3421 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     3481 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     3541 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3601 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     3661 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3721 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3781 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3841 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3901 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3961 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4021 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4081 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4141 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4201 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4261 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4321 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4381 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4441 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4501 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     4561 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     4621 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     4681 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4741 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4801 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     4861 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     4921 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     4981 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     5041 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     5101 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     5161 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     5221 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     5281 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     5341 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     5401 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     5461 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     5521 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     5581 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     5641 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     5701 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     5761 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5821 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     5881 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     5941 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     6001 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     6061 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     6121 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     6181 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     6241 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     6301 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     6361 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     6421 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     6481 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     6541 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     6601 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     6661 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     6721 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     6781 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     6841 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     6901 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     6961 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     7021 aggaatggtg catg
//
