LOCUS       Exported                6480 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses SpyTag003-MBP (maltose binding protein) in bacterial 
            cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    SpyTag003-MBP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6480)
  AUTHORS   Keeble AH, Turkki P, Stokes S, Khairil Anuar INA, Rahikainen R, 
            Hytonen VP, Howarth M
  TITLE     Approaching infinite affinity through engineering of peptide-protein
            interaction.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Dec 10. pii: 1909653116. doi: 
            10.1073/pnas.1909653116.
  PUBMED    31822621
REFERENCE   2  (bases 1 to 6480)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6480
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     191..210
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     294..313
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        294..312
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    313..337
                     /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             352..374
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             393..410
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             420..437
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             504..1601
                     /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     1575..1598
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1638..1655
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1708..1726)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1722..1769
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1806..2261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1893..1912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2103..2124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2353..3168)
                     /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     3076..3095
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3160..3179
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3290..3878
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3779..3798
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4032..4049
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4064..4203
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4129..4148)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4289..4311
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4305..4496)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5305..6387)
                     /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     6349..6368
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6388..6465)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
       61 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
      121 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
      181 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
      241 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
      301 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
      361 ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc
      421 tggtgccgcg cggcagccgt ggcgtgcctc atatcgtgat ggtggacgcc tacaagcgtt
      481 acaagggtag tggtgaaagt ggtaaaatcg aagaaggtaa actggtaatc tggattaacg
      541 gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa gataccggaa
      601 ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag gttgcggcaa
      661 ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc tacgctcaat
      721 ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg tatccgttta
      781 cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct gttgaagcgt
      841 tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc
      901 cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag
      961 aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa
     1021 acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga
     1081 ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat tactccatcg
     1141 cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg tgggcatggt
     1201 ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc ttcaagggtc
     1261 aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca
     1321 aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg
     1381 ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga
     1441 aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc atgccgaaca
     1501 tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg
     1561 gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taattcgagc tcgtaaaagc
     1621 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     1681 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1741 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     1801 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1861 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1921 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1981 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2041 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2101 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2161 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2221 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     2281 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2341 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     2401 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     2461 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     2521 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     2581 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     2641 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     2701 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     2761 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     2821 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     2881 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     2941 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     3001 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     3061 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     3121 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     3181 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     3241 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3301 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3361 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3421 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3481 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3541 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3601 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3661 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3721 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3781 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3841 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3901 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3961 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4021 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     4081 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     4141 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     4201 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4261 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4321 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     4381 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     4441 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     4501 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     4561 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     4621 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     4681 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     4741 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     4801 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     4861 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     4921 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     4981 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     5041 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     5101 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     5161 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     5221 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     5281 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5341 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     5401 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     5461 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     5521 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     5581 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     5641 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     5701 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     5761 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     5821 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     5881 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     5941 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     6001 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     6061 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     6121 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     6181 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     6241 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     6301 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     6361 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     6421 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
//
