LOCUS Exported 6561 bp ds-DNA circular SYN 28-JUL-2021 DEFINITION Expresses AviTag-DogTag-Maltose binding protein in bacterial cytoplasm. The AviTag enables site-specific biotinylation by BirA.. ACCESSION . VERSION . KEYWORDS pET28-AviTag-DogTag-MBP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6561) TITLE DogCatcher allows Loop-friendly Protein-Protein Ligation JOURNAL Unpublished REFERENCE 2 (bases 1 to 6561) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 28, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6561 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(35..1117) /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 1079..1098 /label=LacI-R /note="LacI, reverse primer" promoter complement(1118..1195) /gene="lacI" /label=lacI promoter primer_bind 1401..1420 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1504..1523 /label=T7 /note="T7 promoter, forward primer" promoter 1504..1522 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1523..1547 /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 1562..1584 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1603..1620 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1630..1647 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 1654..1698 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 1795..2892 /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 2866..2889 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 2929..2946 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2999..3017) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3013..3060 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3097..3552 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3184..3203) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3394..3415 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3644..4459) /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 4367..4386 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4451..4470 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4581..5169 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5070..5089 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5323..5340 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5355..5494 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5420..5439) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5580..5602 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5596..5787) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 61 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 121 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 181 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 241 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 301 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 361 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 421 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 481 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 541 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 601 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 661 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 721 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 781 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 841 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 901 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 961 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 1021 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 1081 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 1141 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 1201 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 1261 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 1321 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 1381 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 1441 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 1501 aattaatacg actcactata ggggaattgt gagcggataa caattcccct atagaaataa 1561 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 1621 agcagcggcc tggtgccgcg cggcagccat atgggtctga atgatatttt cgaagcgcag 1681 aaaattgaat ggcatgaagg tagcggagat attccggcta catacgaatt caccgatggt 1741 aaacattata tcaccaatga accgataccg ccgaaaggta gtggtgaaag tggtaaaatc 1801 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa 1861 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa 1921 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg 1981 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac 2041 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag 2101 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg 2161 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa 2221 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct 2281 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc 2341 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa 2401 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca 2461 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat 2521 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg 2581 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac 2641 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta 2701 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa 2761 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc 2821 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa 2881 gacgcgcaga ctaattcgag ctcgtaaaag cttgcggccg cactcgagca ccaccaccac 2941 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc 3001 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg 3061 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa 3121 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 3181 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 3241 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 3301 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 3361 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 3421 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 3481 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 3541 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 3601 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga 3661 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa 3721 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct 3781 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt 3841 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg 3901 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat 3961 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa 4021 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga 4081 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga 4141 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa 4201 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat 4261 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg 4321 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt 4381 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt 4441 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta 4501 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 4561 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 4621 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 4681 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 4741 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 4801 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 4861 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 4921 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 4981 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 5041 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 5101 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 5161 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 5221 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 5281 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 5341 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 5401 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac 5461 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga 5521 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 5581 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg 5641 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc 5701 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt 5761 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat 5821 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga 5881 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga 5941 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc 6001 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga 6061 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca 6121 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt 6181 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat 6241 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt 6301 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag 6361 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag 6421 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac 6481 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat 6541 cggtcgagat cccggtgcct a //