LOCUS Exported 8043 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression of tandem high affinity maltose binding protein domains linked to SpyCatcher, for solid-phase polyprotein synthesis.. ACCESSION . VERSION . KEYWORDS pET21 MBPx-SpyCatcher SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8043) 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 8043) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8043 /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(2783..2800) /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS complement(2810..2827) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" RBS 2847..2869 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind 2884..2908 /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(2908..2927) /label=T7 /note="T7 promoter, forward primer" promoter complement(2909..2927) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3011..3030) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 3236..3313 /gene="lacI" /label=lacI promoter CDS 3314..4396 /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(3333..3352) /label=LacI-R /note="LacI, reverse primer" CDS 5205..5396 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind complement(5390..5412) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 5498..5640 /label=bom /note="basis of mobility region from pBR322" primer_bind 5553..5572 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(5655..5672) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5826..6414) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5906..5925) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6585..7445) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7208..7227 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7446..7550) /gene="bla" /label=AmpR promoter rep_origin complement(7577..8032) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7714..7735) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 7926..7945 /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 gcaagcttct 181 attaaatatg agcgtcacct ttagttgctt tgccatttac agtaacctga ccttgctcat 241 taactgtaaa ggtaatagca gttgctacct cataaccgtc tggtgctgcg gtttcgacaa 301 atgtatattt tcctggatac aggtagaaat ctttcacttg tccatctgaa atccatgtac 361 taatagtttt accagatgaa tcacgcaact ccatagttgc accagctaac tctttgccgt 421 cctcatcacg ttttgagaat ttaatatggg tagcactatc ttcttcaatt gtcatatcac 481 cggactgacc ttgctcactt gataaacctg ataaggtatc aaccatggca ccactaccgc 541 ccgaacccga gctcgaatta gtctgcgcgt ctttcagggc ttcatcgaca gtctgacgac 601 cgctggcggc gttgatcacc gcagtacgca cggcatacca gaaagcggac atctgcggga 661 tgttcggcat gatttcacct ttctgggcgt tttccatagt ggcggccaca cgtggatctt 721 tcaccaactc ttcctcgtaa gacttcagcg ctacggcacc cagcggtttg tctttattaa 781 ccgcttccag accttcatca gtcagcagat agttttcgag gaactctttt gccagctctt 841 tgttcggact ggcggcgtta atacctgcgc tcagcacgcc aacgaacggt ttggatggtt 901 gacccttgaa ggtcggcagt accgttacac cataattcac tttgctggtg tcgatgttgg 961 accatgccca cgggccgttg atggtcatcg ctgtttcgcc tttattaaag gcagcttctg 1021 cgatggagta atcggtgtct gcattcatgt gtttgttttt aatcaggtca accaggaagg 1081 tcagacccgc tttcgcgcca gcgttatcca cgcccacgtc tttaatgtcg ccatacttga 1141 acgcataacc cccgtcagca gcaatcagcg gccaggtgaa gtacggttct tgcaggttga 1201 acatcagcgc gctcttacct ttcgctttca gttctttatc cagcgccggg atctcttccc 1261 aggtttttgg cgggttcggc agcagatctt tgttataaat cagcgataac gcttcaacag 1321 cgatcgggta agcaatcagc ttgccgttgt aacgtacggc atcccaggta aacggataca 1381 gcttgtcctg gaacgctttg tccggggtga tttcagccaa caggccagat tgagcgtagc 1441 caccaaagcg gtcgtgtgcc cagaagataa tgtcagggcc atcgccagtt gccgcaacct 1501 gtgggaattt ctcttccagt ttatccggat gctcaacggt gactttaatt ccggtatctt 1561 tctcgaattt cttaccgact tcagcgagac cgttatagcc tttatcgccg ttaatccaga 1621 ttaccagttt accctcctct atctttccgg atccacctcc ggatccgccg gaactagaat 1681 tcgtctgcgc gtctttcagg gcttcatcga cagtctgacg accgctggcg gcgttgatca 1741 ccgcagtacg cacggcatac cagaaagcgg acatctgcgg gatgttcggc atgatttcac 1801 ctttctgggc gttttccata gtggcggcca cacgtggatc tttcaccaac tcttcctcgt 1861 aagacttcag cgctacggca cccagcggtt tgtctttatt aaccgcttcc agaccttcat 1921 cagtcagcag atagttttcg aggaactctt ttgccagctc tttgttcgga ctggcggcgt 1981 taatacctgc gctcagcacg ccaacgaacg gtttggatgg ttgacccttg aaggtcggca 2041 gtaccgttac accataattc actttgctgg tgtcgatgtt ggaccatgcc cacgggccgt 2101 tgatggtcat cgctgtttcg cctttattaa aggcagcttc tgcgatggag taatcggtgt 2161 ctgcattcat gtgtttgttt ttaatcaggt caaccaggaa ggtcagaccc gctttcgcgc 2221 cagcgttatc cacgcccacg tctttaatgt cgccatactt gaacgcataa cccccgtcag 2281 cagcaatcag cggccaggtg aagtacggtt cttgcaggtt gaacatcagc gcgctcttac 2341 ctttcgcttt cagttcttta tccagcgccg ggatctcttc ccaggttttt ggcgggttcg 2401 gcagcagatc tttgttataa atcagcgata acgcttcaac agcgatcggg taagcaatca 2461 gcttgccgtt gtaacgtacg gcatcccagg taaacggata cagcttgtcc tggaacgctt 2521 tgtccggggt gatttcagcc aacaggccag attgagcgta gccaccaaag cggtcgtgtg 2581 cccagaagat aatgtcaggg ccatcgccag ttgccgcaac ctgtgggaat ttctcttcca 2641 gtttatccgg atgctcaacg gtgactttaa ttccggtatc tttctcgaat ttcttaccga 2701 cttcagcgag accgttatag cctttatcgc cgttaatcca gattaccagt ttaccttctt 2761 cgattttcat gctagccata tggctgccgc gcggcaccag gccgctgctg tgatgatgat 2821 gatgatggct gctgcccata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag 2881 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc 2941 gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg 3001 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg 3061 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg 3121 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta 3181 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac 3241 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt 3301 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc 3361 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga 3421 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact 3481 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc 3541 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt 3601 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc 3661 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt 3721 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat 3781 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc 3841 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct 3901 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg 3961 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg 4021 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc 4081 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac 4141 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct 4201 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa 4261 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac 4321 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 4381 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg 4441 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg 4501 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg 4561 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga 4621 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg 4681 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac 4741 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta 4801 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa 4861 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg 4921 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct 4981 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga 5041 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta 5101 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta 5161 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa 5221 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac 5281 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg 5341 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 5401 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 5461 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 5521 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt 5581 gcaccatata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg 5641 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 5701 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 5761 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 5821 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 5881 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 5941 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 6001 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 6061 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 6121 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 6181 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 6241 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 6301 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 6361 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 6421 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 6481 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 6541 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 6601 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 6661 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 6721 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 6781 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 6841 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 6901 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 6961 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 7021 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 7081 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 7141 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 7201 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 7261 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 7321 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 7381 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 7441 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 7501 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 7561 cgaaaagtgc cacctgaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt 7621 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca 7681 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta 7741 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta 7801 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg 7861 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga 7921 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg 7981 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg 8041 cca //