LOCUS       Exported                5784 bp ds-DNA     circular SYN 22-AUG-2017
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    1454_H14-MBP-SUMO-Cys-Linker-BAF_L58R
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 5784)
  AUTHORS   Matthias Samwer
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Aug 22, 2017 from SnapGene 3.3.4
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5784
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     misc_feature    7..23
                     /note="-43 AT rich region"
     promoter        10..54
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     misc_feature    25..30
                     /note="-35 box"
     misc_feature    30..48
                     /note="lacO"
     protein_bind    30..46
                     /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)."
     misc_feature    48..53
                     /note="-10 box"
     misc_feature    60
                     /note="transcription start"
     misc_feature    62..80
                     /note="lacO"
     protein_bind    62..78
                     /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)."
     misc_feature    96..106
                     /note="RBS"
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     misc_feature    115..187
                     /note="His14-tag"
     misc_feature    193..1300
                     /note="MBP"
     CDS             193..1290
                     /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="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     misc_feature    1324..1611
                     /note="brSUMO"
     misc_feature    1603..1615
                     /note=""
     misc_feature    1616..1692
                     /product="24aa_SG_linker"
                     /label=24aa_SG_linker
     misc_feature    1693..1698
                     /product="AgeI"
                     /label=AgeI
     misc_feature    1694..1969
                     /note="OL with gBlocks"
     misc_feature    1694..1969
                     /note="EcoRI"
     misc_feature    1699..1965
                     /label=BAF
     CDS             1699..1965
                     /codon_start=1
                     /product="BAF_L58R"
                     /translation="MTTSQKHRDFVAEPMGEKPVGSLAGIGEVLGKKLEERGFDKAYVV
                     LGQFLVLKKDEDRFREWLKDTCGANAKQSRDCFGCLREWCDAFL"
     misc_feature    1966..1968
                     /label=Stop
     misc_feature    1969..1974
                     /product="BamHI"
                     /label=BamHI
     CDS             1986..2003
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    2026..2120
                     /note="lambda t0 trx termination region"
     terminator      2026..2120
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     misc_feature    2181..2996
                     /note="KanR"
     CDS             2181..2996
                     /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"
     misc_feature    complement(3209..3306)
                     /note="rrnB T1 trx termination region"
     terminator      3215..3301
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    complement(3354..4455)
                     /note="lacI E.coli genomic sequence"
     CDS             complement(3395..4477)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /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"
     misc_feature    complement(3395..4354)
                     /note="lacI"
     promoter        complement(4478..4555)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     misc_feature    4604..4706
                     /note="part of H-strand Y effector site"
     misc_feature    4747..4887
                     /label=bom
                     /note="basis of mobility region from pBR322"
     misc_feature    5073..5661
                     /note="ColE1 origin"
     rep_origin      complement(5073..5661)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    5074
                     /note="replication origin"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aaccatgagc
      121 aagcatcacc atcattcagg ccatcaccat accggacacc accatcattc aggcagtcat
      181 caccataccg ggaaaactga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
      241 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
      301 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
      361 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
      421 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
      481 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
      541 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
      601 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
      661 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
      721 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
      781 gacctgatta aaaacaaaca catgaatgca gacactgatt actccatcgc agaagctgcc
      841 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
      901 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
      961 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     1021 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     1081 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     1141 attgccgcca ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     1201 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     1261 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct caggtgaaga tgaaagtgga
     1321 ggctctgctg cgggtggcga agaagataag aaaccggcag gtggcgaagg tggcggtgcc
     1381 catatcaacc tgaaagtgaa aggtcaagac ggcaacgaag tctttttccg catcaaacgt
     1441 tctacccagc tgaaaaagct gatgaacgca tactgtgacc gtcagtctgt agacatgacc
     1501 gcaattgctt tcctgtttga tggtcgtcgc ctgcgtgcgg aacagacccc ggatgaactg
     1561 gagatggaag atggcgacga aatcgacgca atgctgcacc agactggtgg cgccgtttgt
     1621 ggaagtggca gtactggttc cggcagctcc ggcagcggcg gcgggagtag cggatcatct
     1681 acagggacca ccaccggtat gacaacctcc caaaagcacc gagacttcgt ggcagagccc
     1741 atgggggaga agccagtggg gagcctggct gggattggtg aagtcctggg caagaagctg
     1801 gaggaaaggg gttttgacaa ggcctatgtt gtccttggcc agtttctggt gctaaagaaa
     1861 gatgaagacc gcttccggga atggctgaaa gacacttgtg gcgccaacgc caagcagtcc
     1921 cgggactgct tcggatgcct tcgagagtgg tgtgatgcct tcttgtaggg atcccagcgg
     1981 ccgcacatca ccatcaccat cactaagctt aattagctga gcttggactc ctgttgatag
     2041 atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg
     2101 cgttttttat tggtgagaat ccaagctagc catgaacaat aaaactgtct gcttacataa
     2161 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc
     2221 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     2281 ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc
     2341 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     2401 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     2461 cttggttact cacgactgcg atccccggca aaacagcatt ccaggtatta gaagaatatc
     2521 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     2581 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
     2641 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     2701 ctgttgaaca agtctggaaa gaaatgcaca aacttttgcc attctcaccg gattcagtcg
     2761 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     2821 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     2881 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     2941 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat
     3001 taattcatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag
     3061 gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag
     3121 tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa
     3181 acgcctgggg taatgactct ctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa
     3241 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     3301 tccgccgctc tagattacgt gcagtcgatg ataagctgtc aaacatgaga attgtgccta
     3361 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3421 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3481 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3541 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3601 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3661 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3721 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3781 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3841 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     3901 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     3961 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4021 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4081 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4141 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4201 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4261 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4321 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4381 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4441 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4501 cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg gtgtcggaat
     4561 ttcgggcagc gttgggtcct ggccacgggt gcgcatgatc tagagctgcc tcgcgcgttt
     4621 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     4681 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     4741 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     4801 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     4861 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     4921 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4981 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5041 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5101 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5161 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5221 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5281 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5341 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5401 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5461 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     5521 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5581 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5641 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5701 tggaacgaaa actcacgtta agggattttg gtcatgacat taacctataa aaataggcgt
     5761 atcacgaggc cctttcgtct tcac
//
