LOCUS       MBP-(TEV)-SA_addgene-RG 6305 bp ds-DNA     circular     21-JAN-2024
DEFINITION  .
FEATURES             Location/Qualifiers
     primer_bind     861..880
                     /label="LacI-R"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="LacI, reverse primer"
     promoter        complement(900..977)
                     /label="lacI promoter"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
                     /gene="lacI"
     primer_bind     1183..1202
                     /label="pBRrevBam"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
                     /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer"
     promoter        1286..1304
                     /label="T7 promoter"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1286..1305
                     /label="T7"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /note="T7 promoter, forward primer"
     protein_bind    1305..1329
                     /label="lac operator"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /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)."
                     /bound_moiety="lac repressor encoded by lacI"
     RBS             1344..1366
                     /label="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
     CDS             1377..1394
                     /label="6xHis"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /product="6xHis affinity tag"
                     /translation="HHHHHH"
     regulatory      1391..1400
                     /label="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
                     /regulatory_class="other"
     CDS             1407..2504
                     /label="MBP"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol."
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQ"
     primer_bind     2481..2504
                     /label="MBP-F"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="Maltose binding protein, forward primer"
     CDS             2505..2552
                     /label="linker"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="NSSSNNNNNNNNNNLG"
     CDS             2553..2573
                     /label="TEV site"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /product="tobacco etch virus (TEV) protease recognition andcleavage site"
                     /translation="ENLYFQG"
     CDS             2574..2957
                     /label="SA"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS"
     CDS             2958..2975
                     /label="thrombin site"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /product="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     misc_feature    2979..3479
                     /label="RGG"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     CDS             2979..3479
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="ESNQSNNGGSGNAALNRGGRYVPPHLRGGDGGAAAAASAGGDDRRGGAGGGGYRRGGGNSGGGGGGGYDRGYNDNRDDRDNRGGSGGYGRDRNYEDRGYNGGGGGGGNRGYNNNRGGGGGGYNRQDRGDGGSSNFSRGGYNNRDEGSDNRGSGRSYNNDRRDNGGDG"
     CDS             3486..3503
                     /label="6xHis"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /product="6xHis affinity tag"
                     /translation="HHHHHH"
     primer_bind     complement(3556..3574)
                     /label="T7 Term"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="T7 terminator, reverse primer"
     terminator      3570..3617
                     /label="T7 terminator"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="transcription terminator for bacteriophage T7 RNA polymerase"
     rep_origin      3654..4109
                     /label="f1 ori"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
                     /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
                     /direction="RIGHT"
     primer_bind     complement(3741..3760)
                     /label="F1ori-R"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="F1 origin, reverse primer"
     primer_bind     3951..3972
                     /label="F1ori-F"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /note="F1 origin, forward primer"
     CDS             complement(4224..5039)
                     /label="KanR"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes"
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     primer_bind     4947..4966
                     /label="Kan-R"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5031..5050
                     /label="pENTR-R"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
                     /note="pENTR vectors, reverse primer"
     rep_origin      5192..5780
                     /label="ori"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
                     /direction="RIGHT"
     primer_bind     5681..5700
                     /label="pBR322ori-F"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
                     /note="pBR322 origin, forward primer"
     primer_bind     5934..5951
                     /label="L4440"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
                     /note="L4440 vector, forward primer"
     CDS             complement(6122..899)
                     /label="lacI"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /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)."
                     /gene="lacI"
                     /product="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"
ORIGIN
        1 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
       61 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
      121 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
      181 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
      241 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
      301 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
      361 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
      421 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
      481 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
      541 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
      601 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
      661 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
      721 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
      781 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
      841 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
      901 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
      961 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     1021 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     1081 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     1141 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     1201 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     1261 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     1321 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatgcacc
     1381 atcaccatca ccatggcggc ggatctatga aaatcgaaga aggtaaactg gtaatctgga
     1441 ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata
     1501 ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg
     1561 cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg
     1621 ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc
     1681 cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg
     1741 aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag
     1801 agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc
     1861 tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt
     1921 atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg
     1981 gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact
     2041 ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg
     2101 catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca
     2161 agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc
     2221 cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg
     2281 aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt
     2341 tggtgaaaga tccgcgtatt gccgccacta tggaaaacgc ccagaaaggt gaaatcatgc
     2401 cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg
     2461 ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagaattcg agctcgaaca
     2521 acaacaacaa taacaataac aacaacctcg gagagaacct gtatttccag ggcatggctg
     2581 aagctggtat caccggcacc tggtacaacc agctgggatc caccttcatc gttaccgctg
     2641 gtgctgacgg tgctctgacc ggtacctacg aatccgctgt tggtaacgct gaatctagat
     2701 acgttctgac cggtcgttac gactccgctc cggctaccga cggttccgga accgctctgg
     2761 gttggaccgt tgcttggaaa aacaactacc gtaacgctca ctccgctacc acctggtctg
     2821 gccagtacgt tggtggtgct gaagctcgta tcaacaccca gtggttgttg acctccggca
     2881 ccaccgaagc caacgcgtgg aaatccaccc tggttggtca cgacaccttc accaaagtta
     2941 aaccgtccgc tgcttcctta gtacctcgcg ggtcaatgga atccaatcaa tccaataacg
     3001 gtggctcggg taatgccgct ctgaatcgtg gtggccgcta cgtgccgccg catctgcgtg
     3061 gtggcgatgg tggtgcagca gcagctgcat ctgctggcgg tgatgaccgt cgcggcggtg
     3121 cgggcggtgg cggttatcgt cgcggcggtg gcaatagtgg tggcggtggc ggtggcggtt
     3181 atgatcgtgg ttacaatgac aaccgtgatg accgcgataa ccgtggcggt tccggcggtt
     3241 acggtcgtga tcgtaattat gaagaccgtg gttacaacgg cggtggcggt ggcggtggca
     3301 atcgtggcta taacaataac cgtggtggcg gtggcggtgg ctacaatcgc caggatcgtg
     3361 gtgacggtgg cagctctaac ttttcgcgcg gtggctataa taaccgtgat gaaggcagcg
     3421 acaaccgcgg tagtggccgt tcctacaata acgatcgtcg cgacaatggt ggcgatggcc
     3481 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     3541 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     3601 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     3661 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3721 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3781 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3841 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3901 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3961 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4021 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4081 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggtac ccaggtggca
     4141 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4201 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     4261 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     4321 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     4381 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     4441 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     4501 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     4561 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     4621 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     4681 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     4741 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     4801 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     4861 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     4921 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     4981 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     5041 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     5101 ttaacgtgac ctaggtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5161 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5221 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5281 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     5341 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5401 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5461 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5521 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5581 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5641 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5701 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5761 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5821 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5881 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5941 cagtgagcga ggaagcggaa gagcgccata atccatgata aagaagacag tcataagtgc
     6001 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     6061 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     6121 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6181 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     6241 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     6301 ccacg
//