LOCUS       Exported                8021 bp ds-DNA     circular SYN 31-JAN-2023
DEFINITION  Expresses MBP-GSK3-beta_S9A-HA-His (human Glycogen Synthase Kinase 
            3-beta S9A mutant as a fusion protein with MBP, HA, and His- tags) 
            in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    pES002 MBP-GSK3-beta_S9A-HA-His
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8021)
  AUTHORS   Gavagan M, Jameson N, Zalatan JG
  TITLE     The Axin scaffold protects the kinase GSK3-beta from cross-pathway 
            inhibition
  JOURNAL   bioRxiv 2022.12.05.519208
REFERENCE   2  (bases 1 to 8021)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 31, 2023 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 ccaccgcctt 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
//
