LOCUS       Exported                6481 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression of Maltose Binding Protein linked to SnoopTag, 
            a peptide tag forming a spontaneous covalent bond with its protein 
            partner Snoopcatcher..
ACCESSION   .
VERSION     .
KEYWORDS    pET28a SnoopTag-MBP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6481)
  AUTHORS   Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, 
            Howarth M
  TITLE     Programmable polyproteams built using twin peptide superglues.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Jan 19. pii: 201519214.
  PUBMED    26787909
REFERENCE   2  (bases 1 to 6481)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6481
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      26..73
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(194..1291)
                     /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"
     primer_bind     complement(197..220)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(1355..1372)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1382..1399)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1418..1440
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1455..1479
                     /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     complement(1479..1498)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1480..1498)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1582..1601)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1807..1884
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1885..2967
                     /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(1904..1923)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3776..3967
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3961..3983)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4069..4211
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4124..4143
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4226..4243)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4397..4985)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4477..4496)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5096..5115)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5107..5922
                     /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     complement(5180..5199)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6015..6470)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6152..6173)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6364..6383
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagctttt
      181 acgagctcga attagtctgc gcgtctttca gggcttcatc gacagtctga cgaccgctgg
      241 cggcgttgat caccgcagta cgcacggcat accagaaagc ggacatctgc gggatgttcg
      301 gcatgatttc acctttctgg gcgttttcca tagtggcggc aatacgtgga tctttcgcca
      361 actcttcctc gtaagacttc agcgctacgg cacccagcgg tttgtcttta ttaaccgctt
      421 ccagaccttc atcagtcagc agatagtttt cgaggaactc ttttgccagc tctttgttcg
      481 gactggcggc gttaatacct gcgctcagca cgccaacgaa cggtttggat ggttgaccct
      541 tgaaggtcgg cagtaccgtt acaccataat tcactttgct ggtgtcgatg ttggaccatg
      601 cccacgggcc gttgatggtc atcgctgttt cgcctttatt aaaggcagct tctgcgatgg
      661 agtaatcggt gtctgcattc atgtgtttgt ttttaatcag gtcaaccagg aaggtcagac
      721 ccgctttcgc gccagcgtta tccacgccca cgtctttaat gtcgtacttg ccgttttcat
      781 acttgaacgc ataacccccg tcagcagcaa tcagcggcca ggtgaagtac ggttcttgca
      841 ggttgaacat cagcgcgctc ttacctttcg ctttcagttc tttatccagc gccgggatct
      901 cttcccaggt ttttggcggg ttcggcagca gatctttgtt ataaatcagc gataacgctt
      961 caacagcgat cgggtaagca atcagcttgc cgttgtaacg tacggcatcc caggtaaacg
     1021 gatacagctt gtcctggaac gctttgtccg gggtgatttc agccaacagg ccagattgag
     1081 cgtagccacc aaagcggtcg tgtgcccaga agataatgtc agggccatcg ccagttgccg
     1141 caacctgtgg gaatttctct tccagtttat ccggatgctc aacggtgact ttaattccgg
     1201 tatctttctc gaatttctta ccgacttcag cgagaccgtt atagccttta tcgccgttaa
     1261 tccagattac cagtttacct tcttcgattt taccactttc accactacct ttgttcactt
     1321 taataaattc aatatcgccc agttttccca tatggctgcc gcgcggcacc aggccgctgc
     1381 tgtgatgatg atgatgatgg ctgctgccca tggtatatct ccttcttaaa gttaaacaaa
     1441 attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat
     1501 ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg
     1561 ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc
     1621 gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg
     1681 ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg
     1741 gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag
     1801 atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     1861 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     1921 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     1981 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     2041 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     2101 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     2161 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     2221 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     2281 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     2341 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     2401 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     2461 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     2521 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     2581 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     2641 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     2701 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     2761 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     2821 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     2881 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     2941 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     3001 ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     3061 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     3121 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
     3181 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
     3241 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
     3301 ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
     3361 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
     3421 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
     3481 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
     3541 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
     3601 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
     3661 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
     3721 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
     3781 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
     3841 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
     3901 cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa
     3961 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     4021 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     4081 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     4141 gtactgagag tgcaccatat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     4201 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4261 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4321 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4381 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4441 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4501 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4561 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4621 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4681 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4741 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4801 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     4861 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4921 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4981 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5041 gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     5101 ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac
     5161 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     5221 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     5281 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     5341 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     5401 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     5461 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     5521 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     5581 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     5641 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     5701 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     5761 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     5821 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     5881 aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg
     5941 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6001 aaaagtgcca cctgaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg
     6061 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     6121 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     6181 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     6241 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     6301 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     6361 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     6421 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     6481 a
//
