LOCUS       Exported                7336 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  MBP-rPKL WT for production and purification of rat PKL WT.
ACCESSION   .
VERSION     .
KEYWORDS    MBP-rPKL WT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7336)
  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 7336)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7336
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(239..1117)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1144..1428
                     /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     1351..1370
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1462..2562
                     /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     2536..2559
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2611..2622
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4255..4272
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4320..4337)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4490..4576
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4668..4695
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4771..4789)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4785..4832
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4929..5384
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5016..5035)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5226..5247
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5410..5514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5515..6375
                     /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(5733..5752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6546..7134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7035..7054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7288..7305
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcatg gggagacccc
       61 acactaccat cggcgctacg gcgtttcact tctgagttcg gcatggggtc aggtgggacc
      121 accgcgctac tgccgccagg catctagctg cataatgtgc ctgtcaaatg gacgaagcag
      181 ggattctgca aaccctatgc tactccgtca agccgtcaat tgtctgattc gttaccaatt
      241 atgacaactt gacggctaca tcattcactt tttcttcaca accggcacgg aactcgctcg
      301 ggctggcccc ggtgcatttt ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa
      361 cattgcgacc gacggtggcg ataggcatcc gggtggtgct caaaagcagc ttcgcctggc
      421 tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc taactgctgg cggaaaagat
      481 gtgacagacg cgacggcgac aagcaaacat gctgtgcgac gctggcgata tcaaaattgc
      541 tgtctgccag gtgatcgctg atgtactgac aagcctcgcg tacccgatta tccatcggtg
      601 gatggagcga ctcgttaatc gcttccatgc gccgcagtaa caattgctca agcagattta
      661 tcgccagcag ctccgaatag cgcccttccc cttgcccggc gttaatgatt tgcccaaaca
      721 ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata
      781 ttgacggcca gttaagccat tcatgccagt aggcgcgcgg acgaaagtaa acccactggt
      841 gataccattc gcgagcctcc ggatgacgac cgtagtgatg aatctctcct ggcgggaaca
      901 gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg atttttcacc accccctgac
      961 cgcgaatggt gagattgaga atataacctt tcattcccag cggtcggtcg ataaaaaaat
     1021 cgagataacc gttggcctca atcggcgtta aacccgccac cagatgggca ttaaacgagt
     1081 atcccggcag caggggatca ttttgcgctt cagccatact tttcatactc ccgccattca
     1141 gagaagaaac caattgtcca tattgcatca gacattgccg tcactgcgtc ttttactggc
     1201 tcttctcgct aaccaaaccg gtaaccccgc ttattaaaag cattctgtaa caaagcggga
     1261 ccaaagccat gacaaaaacg cgtaacaaaa gtgtctataa tcacggcaga aaagtccaca
     1321 ttgattattt gcacggcgtc acactttgct atgccatagc atttttatcc ataagattag
     1381 cggatcctac ctgacgcttt ttatcgcaac tctctactgt ttctccatac ccgttttttg
     1441 ggctaacagg aggaattaca tatgaaaatc gaagaaggta aactggtaat ctggattaac
     1501 ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga
     1561 attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca
     1621 actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg ctacgctcaa
     1681 tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct gtatccgttt
     1741 acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc tgttgaagcg
     1801 ttatcgctga tttataacaa ggatctgctg ccgaacccgc caaaaacctg ggaagagatc
     1861 ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa
     1921 gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa
     1981 aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa agcgggtctg
     2041 accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc
     2101 gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg
     2161 tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac cttcaagggt
     2221 caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac
     2281 aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg tctggaagcg
     2341 gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggcg
     2401 aaagatccac gtattgccgc cactatggaa aacgcccaga aaggtgaaat catgccgaac
     2461 atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc
     2521 ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac
     2581 aacaacaata acaataacaa caacctcggg atcgagggaa ggcatatgga aggtccggca
     2641 ggttacctgc gccgtgcgag cgttgcgcag ctgacccaag aactgggcac cgcattcttc
     2701 cagcaacaac agctgccggc ggctatggct gacacgttcc tggaacacct gtgcctgctg
     2761 gatattgatt cccagccagt ggcggctcgc agcacttcca ttatcgctac cattggtcca
     2821 gcttcccgtt ctgtcgatcg cctgaaagaa atgatcaaag caggcatgaa catcgctcgt
     2881 ctgaactttt ctcacggttc tcacgaatac cacgctgaaa gcatcgcgaa tatccgtgaa
     2941 gctaccgaat ccttcgcgac ctccccgctg agctaccgtc cggtagcaat cgcgctggac
     3001 accaaaggcc cggaaattcg taccggcgtg ctgcagggtg gtccggaatc cgaagttgaa
     3061 attgtgaaag gttctcaggt gctggtgacc gttgacccga aattccaaac gcgtggcgat
     3121 gcaaaaactg tgtgggtaga ctaccacaac attactcgtg tggtagcggt aggtggccgt
     3181 atctacatcg atgatggcct gatctctctg gtggttcaga aaatcggtcc ggaaggtctg
     3241 gttaccgaag ttgagcacgg tggtatcctg ggttctcgta aaggcgttaa cctgcctaac
     3301 acggaagttg acctgccggg cctgagcgaa caggatctgc tggatctgcg tttcggcgtt
     3361 caacacaacg tggatattat cttcgcatcc ttcgttcgta aagcgtctga cgtgctggct
     3421 gtacgtgatg cgctgggtcc agaaggtcag aatatcaaga tcatctctaa aattgaaaat
     3481 cacgaaggcg tgaaaaaatt tgacgaaatc ctggaagttt ccgatggcat tatggtggcg
     3541 cgcggcgacc tgggcattga aattccggcc gagaaagttt tcctggctca aaagatgatg
     3601 atcggtcgct gtaacctggc tggcaagccg gtagtatgcg cgacccagat gctggagagc
     3661 atgatcacta aagcccgccc aactcgcgcc gaaaccagcg atgttgcaaa cgctgtgctg
     3721 gacggtgcag attgcattat gctgtctggc gaaaccgcaa aaggttcttt tccggttgaa
     3781 gctgttatga tgcaacacgc aatcgcgcgt gaagctgaag cggccgtgta ccaccgccag
     3841 ctgttcgaag aactgcgccg tgctgcaccg ctgtctcgcg acccgactga agttaccgct
     3901 atcggcgcag ttgaggcttc ctttaaatgt tgcgccgccg ctattatcgt cctgactaaa
     3961 accggccgct ccgcccaact gctgtctcag tatcgcccac gcgctgctgt tatcgccgtt
     4021 acgcgcagcg cgcaggcagc ccgtcaggtt cacctgtctc gtggcgtttt cccgctgctg
     4081 taccgtgaac cgcctgaagc gatctgggcc gacgatgtcg atcgtcgtgt tcagtttggt
     4141 attgaatccg gcaaactgcg tggtttcctg cgtgttggtg acctggttat cgtcgttact
     4201 ggttggcgtc cgggttctgg ttataccaac attatgcgcg ttctgtctgt gtcccaccac
     4261 catcaccacc actaagcttg agctcttggc tgttttggcg gatgagagaa gattttcagc
     4321 ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc
     4381 agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc
     4441 gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg
     4501 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     4561 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg
     4621 gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct
     4681 gacggatggc ctttttgcgt ttctacaaac tcctcgagct tgatccggct gctaacaaag
     4741 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     4801 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatctg
     4861 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     4921 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4981 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5041 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5101 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5161 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5221 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5281 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5341 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5401 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5461 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5521 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5581 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5641 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5701 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5761 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5821 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5881 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5941 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6001 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6061 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6121 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6181 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6241 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6301 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6361 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6421 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6481 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6541 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6601 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6661 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6721 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6781 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6841 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6901 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6961 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7021 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7081 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7141 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7201 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7261 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc caaagggccc
     7321 aaggcccagt ctttcg
//
