LOCUS Exported 8021 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses MBP-GSK3-beta-HA-His (human Glycogen Synthase Kinase 3-beta as a fusion protein with MBP, HA, and His- tags) in E.coli. ACCESSION . VERSION . KEYWORDS pES001 MBP-GSK3-beta-HA-His SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8021) AUTHORS Gavagan M, Fagnan E, Speltz EB, Zalatan JG TITLE The Scaffold Protein Axin Promotes Signaling Specificity within the Wnt Pathway by Suppressing Competing Kinase Reactions. JOURNAL Cell Syst. 2020 Jun 24;10(6):515-525.e5. doi: 10.1016/j.cels.2020.05.002. Epub 2020 Jun 17. PUBMED 32553184 REFERENCE 2 (bases 1 to 8021) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8021 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 3..80 /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." CDS 81..1163 /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(100..119) /label=LacI-R /note="LacI, reverse primer" promoter 1406..1434 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 1442..1458 /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 1447..1469 /label=M13/pUC Reverse /note="In lacZ gene" CDS 1528..2628 /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="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" regulatory 2485..2494 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 2602..2625 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 2677..2688 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" CDS 2731..2751 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3569..3580 /codon_start=1 /product="WELQut protease recognition and cleavage site" /label=WELQut site /translation="WELQ" CDS 4015..4041 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 4069..4086 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4113..4130) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4113..4129) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4122..4144) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(4308..4325) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 4478..4564 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4656..4683 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" promoter 4703..4794 /gene="bla" /label=AmpR promoter CDS 4795..5655 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5013..5032) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin complement(5697..6210) /direction=LEFT /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5892..5913) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 6104..6123 /label=F1ori-R /note="F1 origin, reverse primer" rep_origin 6321..6909 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6810..6829 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7063..7080 /label=L4440 /note="L4440 vector, forward primer" misc_feature 7095..7235 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(7161..7180) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7321..7343 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(7337..7528) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga 61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg 121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa 181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac 241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc 301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg 361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc 421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca 481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga 541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc 601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg 661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag 721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga 781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa 841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg 901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc 961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata 1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag 1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg 1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga 1501 gcacttcacc aacaaggacc atagattatg aaaatcgaag aaggtaaact ggtaatctgg 1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat 1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt 1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac 1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat 1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt 1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa 1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac 1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag 2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg 2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac 2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg 2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc 2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt 2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg 2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag 2461 ttggcgaaag atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg 2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc 2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg 2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat ttcaactagt 2701 ggatctggtg gtggtggcgg ttccatgagc gagaatcttt attttcaggg atcctcaggg 2761 cggcccagaa ccacctcctt tgcggagagc tgcaagccgg tgcagcagcc ttcagctttt 2821 ggcagcatga aagttagcag agacaaggac ggcagcaagg tgacaacagt ggtggcaact 2881 cctgggcagg gtccagacag gccacaagaa gtcagctata cagacactaa agtgattgga 2941 aatggatcat ttggtgtggt atatcaagcc aaactttgtg attcaggaga actggtcgcc 3001 atcaagaaag tattgcagga caagagattt aagaatcgag agctccagat catgagaaag 3061 ctagatcact gtaacatagt ccgattgcgt tatttcttct actccagtgg tgagaagaaa 3121 gatgaggtct atcttaatct ggtgctggac tatgttccgg aaacagtata cagagttgcc 3181 agacactata gtcgagccaa acagacgctc cctgtgattt atgtcaagtt gtatatgtat 3241 cagctgttcc gaagtttagc ctatatccat tcctttggaa tctgccatcg ggatattaaa 3301 ccgcagaacc tcttgttgga tcctgatact gctgtattaa aactctgtga ctttggaagt 3361 gcaaagcagc tggtccgagg agaacccaat gtttcgtata tctgttctcg gtactatagg 3421 gcaccagagt tgatctttgg agccactgat tatacctcta gtatagatgt atggtctgct 3481 ggctgtgtgt tggctgagct gttactagga caaccaatat ttccagggga tagtggtgtg 3541 gatcagttgg tagaaataat caaggtcctg ggaactccaa caagggagca aatcagagaa 3601 atgaacccaa actacacaga atttaaattc cctcaaatta aggcacatcc ttggactaag 3661 gtcttccgac cccgaactcc accggaggca attgcactgt gtagccgtct gctggagtat 3721 acaccaactg cccgactaac accactggaa gcttgtgcac attcattttt tgatgaatta 3781 cgggacccaa atgtcaaact accaaatggg cgagacacac ctgcactctt caacttcacc 3841 actcaagaac tgtcaagtaa tccacctctg gctaccatcc ttattcctcc tcatgctcgg 3901 attcaagcag ctgcttcaac ccccacaaat gccacagcag cgtcagatgc taatactgga 3961 gaccgtggac agaccaataa tgctgcttct gcatcagctt ccaactccac tagttaccca 4021 tacgatgttc cagattacgc aagcttgggt ggtcccaaca gcggccgcca ccatcaccac 4081 caccactagg ggccgcactc gagaagcttg gcactggccg tcgttttaca acgtcgtgac 4141 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 4201 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 4261 ggcgaatggc agcttggctg ttttggcgga tgagataaga ttttcagcct gatacagatt 4321 aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg 4381 gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg 4441 gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc 4501 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac 4561 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg 4621 acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct 4681 ttttgcgttt ctacaaactc tttttgttta tttttctaaa tacattcaaa tatgtatccg 4741 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 4801 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 4861 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 4921 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 4981 cgttctccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt 5041 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 5101 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 5161 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 5221 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 5281 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 5341 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 5401 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 5461 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 5521 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 5581 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 5641 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaccc 5701 cggttgataa tcagaaaagc cccaaaaaca ggaagattgt ataagcaaat atttaaattg 5761 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 5821 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagggt 5881 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 5941 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca cccaaatcaa 6001 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat 6061 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag 6121 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg 6181 ccgcgcttaa tgcgccgcta cagggcgcgt aaaaggatct aggtgaagat cctttttgat 6241 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 6301 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 6361 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 6421 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 6481 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 6541 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 6601 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 6661 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 6721 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 6781 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 6841 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 6901 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 6961 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 7021 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 7081 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 7141 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca 7201 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg 7261 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 7321 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc 7381 ggtaaagctc atcagcgtgg tcgtgcagcg attcacagat gtctgcctgt tcatccgcgt 7441 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt 7501 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca 7561 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg 7621 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg 7681 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc 7741 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg 7801 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc 7861 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat 7921 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga 7981 tcatgcgcac ccgtggccag gacccaacgc tgcccgaaat t //