LOCUS       Exported                7300 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  MBP-rPKM2 WT for production and purification of rat PKM2 WT.
ACCESSION   .
VERSION     .
KEYWORDS    MBP-rPKM2 WT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7300)
  AUTHORS   Gassaway BM, Cardone RL, Padyana AK, Petersen MC, Judd ET, Hayes S, 
            Tong S, Barber KW, Apostolidi M, Abulizi A, Sheetz JB, Kshitiz, 
            Aerni HR, Gross S, Kung C, Samuel VT, Shulman GI, Kibbey RG, 
            Rinehart J
  TITLE     Distinct Hepatic PKA and CDK Signaling Pathways Control 
            Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic 
            Glucose Production.
  JOURNAL   Cell Rep. 2019 Dec 10;29(11):3394-3404.e9. doi: 
            10.1016/j.celrep.2019.11.009.
  PUBMED    31825824
REFERENCE   2  (bases 1 to 7300)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7300
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(229..1107)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1134..1418
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1341..1360
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1452..2552
                     /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"
     primer_bind     2526..2549
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2601..2612
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4209..4226
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4274..4291)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4444..4530
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4622..4649
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4725..4743)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4739..4786
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4883..5338
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4970..4989)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5180..5201
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5364..5468
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5469..6329
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5687..5706)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6500..7088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6989..7008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7242..7259
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcgttttatt tgatgcctgg cagttcccta ctctcgcatg gggagacccc acactaccat
       61 cggcgctacg gcgtttcact tctgagttcg gcatggggtc aggtgggacc accgcgctac
      121 tgccgccagg catctagctg cataatgtgc ctgtcaaatg gacgaagcag ggattctgca
      181 aaccctatgc tactccgtca agccgtcaat tgtctgattc gttaccaatt atgacaactt
      241 gacggctaca tcattcactt tttcttcaca accggcacgg aactcgctcg ggctggcccc
      301 ggtgcatttt ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc
      361 gacggtggcg ataggcatcc gggtggtgct caaaagcagc ttcgcctggc tgatacgttg
      421 gtcctcgcgc cagcttaaga cgctaatccc taactgctgg cggaaaagat gtgacagacg
      481 cgacggcgac aagcaaacat gctgtgcgac gctggcgata tcaaaattgc tgtctgccag
      541 gtgatcgctg atgtactgac aagcctcgcg tacccgatta tccatcggtg gatggagcga
      601 ctcgttaatc gcttccatgc gccgcagtaa caattgctca agcagattta tcgccagcag
      661 ctccgaatag cgcccttccc cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa
      721 atgcggctgg tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca
      781 gttaagccat tcatgccagt aggcgcgcgg acgaaagtaa acccactggt gataccattc
      841 gcgagcctcc ggatgacgac cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc
      901 acccggtcgg caaacaaatt ctcgtccctg atttttcacc accccctgac cgcgaatggt
      961 gagattgaga atataacctt tcattcccag cggtcggtcg ataaaaaaat cgagataacc
     1021 gttggcctca atcggcgtta aacccgccac cagatgggca ttaaacgagt atcccggcag
     1081 caggggatca ttttgcgctt cagccatact tttcatactc ccgccattca gagaagaaac
     1141 caattgtcca tattgcatca gacattgccg tcactgcgtc ttttactggc tcttctcgct
     1201 aaccaaaccg gtaaccccgc ttattaaaag cattctgtaa caaagcggga ccaaagccat
     1261 gacaaaaacg cgtaacaaaa gtgtctataa tcacggcaga aaagtccaca ttgattattt
     1321 gcacggcgtc acactttgct atgccatagc atttttatcc ataagattag cggatcctac
     1381 ctgacgcttt ttatcgcaac tctctactgt ttctccatac ccgttttttg ggctaacagg
     1441 aggaattaca tatgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag
     1501 gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca
     1561 ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg
     1621 gccctgacat tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt
     1681 tggctgaaat caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg
     1741 ccgtacgtta caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga
     1801 tttataacaa ggatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg
     1861 ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact
     1921 tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt
     1981 acgacattaa agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg
     2041 ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg
     2101 cctttaataa aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg
     2161 acaccagcaa agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca
     2221 aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg
     2281 caaaagagtt cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag
     2341 acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac
     2401 gtattgccgc cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga
     2461 tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga
     2521 ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac aacaacaata
     2581 acaataacaa caacctcggg atcgagggaa ggcatatgcc gaagccagac tctgaggccg
     2641 gaacagcctt catccaaaca cagcaacttc atgctgctat ggctgatact ttcttagaac
     2701 acatgtgtcg tttggacatt gactccgcgc ctatcacggc tcgcaatacc ggaatcatct
     2761 gtacgatcgg ccctgcgagt cgctcggttg aaatgttaaa ggagatgatt aagtcgggca
     2821 tgaacgttgc acgtttaaat ttctcacatg gtacacacga atatcacgca gagacgatca
     2881 aaaacgttcg cgctgctacg gagtcctttg catcagatcc tatcttatat cgcccagtag
     2941 cagtcgcact ggataccaaa gggcctgaga ttcgcaccgg gctgatcaag ggctctggaa
     3001 ccgctgaagt tgagcttaag aaaggagcga cattaaaaat caccttagac aacgcgtaca
     3061 tggagaaatg cgacgaaaac attttatggt tggactataa gaatatctgt aaagttgttg
     3121 aggtaggttc gaagatttat gtcgacgacg gcttaatctc cctgcaagta aaggagaaag
     3181 gagcggatta tttagtaaca gaagtagaaa atggtggctc cctgggatcg aagaaaggag
     3241 tgaatttgcc aggcgcggcg gttgacctgc cagcagtatc tgagaaagac atccaagacc
     3301 tgaagttcgg tgtggagcag gacgtagaca tggtgttcgc ttcgtttatt cgtaaagcgg
     3361 ctgatgtcca cgaggtgcgc aaagttcttg gagaaaaggg caagaatatt aaaatcattt
     3421 ccaaaatcga aaatcacgag ggcgtccgtc gttttgacga gattttggaa gctagcgacg
     3481 gtattatggt ggctcgtggc gacctgggaa ttgaaattcc cgccgagaag gtcttcctgg
     3541 cgcagaagat gatgatcggg cgctgcaacc gcgcggggaa gccggtaatt tgtgcgaccc
     3601 aaatgcttga gagtatgatt aagaaaccgc gtccaacgcg tgctgagggt agcgatgttg
     3661 ctaatgcagt attagacggt gccgactgta ttatgttaag tggtgagact gctaaagggg
     3721 attatcccct tgaagctgta cgcatgcagc atttaattgc tcgcgaggca gaggcagcaa
     3781 tctaccacct tcaattgttt gaggaactgc gccgccttgc gcccatcaca agtgacccta
     3841 ccgaagctgc tgcggtggga gcagttgagg cgagttttaa atgctgttct ggcgccatca
     3901 tcgtacttac taagagcggt cgctccgcac accaggttgc gcgctatcgt ccccgtgcac
     3961 ccatcattgc agtaacacgt aatccccaga cagctcgcca agcacatctg tatcgcggca
     4021 tctttcccgt cttgtgtaaa gatgcggtac ttgacgcctg ggctgaagat gttgaccttc
     4081 gcgtaaactt ggctatgaat gtcggaaaag cccgtgggtt cttcaaaaag ggggacgtag
     4141 tcatcgttct tacgggctgg cgtcccggtt ctgggtttac caatacaatg cgcgtagtcc
     4201 cggtcccaca ccaccatcac caccactaag cttgagctct tggctgtttt ggcggatgag
     4261 agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga
     4321 atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga
     4381 aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg
     4441 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     4501 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag
     4561 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag
     4621 cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactcctcg agcttgatcc
     4681 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     4741 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     4801 tatatccgga tctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     4861 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4921 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     4981 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5041 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5101 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5161 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5221 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5281 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     5341 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5401 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5461 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5521 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5581 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5641 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5701 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5761 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5821 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5881 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5941 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6001 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6061 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6121 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6181 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6241 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6301 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6361 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6421 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6481 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6541 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6601 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6661 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6721 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6781 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6841 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6901 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6961 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7021 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7081 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7141 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     7201 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     7261 aagccaaagg gcccaaggcc cagtctttcg actgagcctt
//
