LOCUS       sequence-204878-        7336 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MBP-rPKL construct for production and purification of rat PKL pS12.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2019.11"; journalName: "Cell Rep"; date: 
            "2019-12-10- 10"; volume: "29"; issue: "11"; pages: "3394-3404.e9"
COMMENT     Sequence Label: MBP-rPKL S12TAG
FEATURES             Location/Qualifiers
     source          1..7336
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(245..1123)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1150..1434
                     /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     1357..1376
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1468..2568
                     /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     2542..2565
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2617..2628
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4261..4278
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4326..4343)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4496..4582
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4674..4701
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4777..4795)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4791..4838
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4935..5390
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5022..5041)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5232..5253
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5416..5520
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5521..5589,5590..6381)
                     /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(5739..5758)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6552..7140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7041..7060
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7294..7311
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctttcgactg agcctttcgt tttatttgat gcctggcagt tccctactct cgcatgggga
       61 gaccccacac taccatcggc gctacggcgt ttcacttctg agttcggcat ggggtcaggt
      121 gggaccaccg cgctactgcc gccaggcatc tagctgcata atgtgcctgt caaatggacg
      181 aagcagggat tctgcaaacc ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta
      241 ccaattatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
      301 cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
      361 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
      421 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
      481 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa
      541 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
      601 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
      661 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
      721 caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg
      781 caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
      841 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
      901 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
      961 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
     1021 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
     1081 acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccgc
     1141 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
     1201 actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
     1261 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
     1321 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
     1381 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt
     1441 tttttgggct aacaggagga attacatatg aaaatcgaag aaggtaaact ggtaatctgg
     1501 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     1561 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     1621 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     1681 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     1741 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     1801 gaagcgttat cgctgattta taacaaggat ctgctgccga acccgccaaa aacctgggaa
     1861 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     1921 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     1981 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     2041 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     2101 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     2161 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     2221 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     2281 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     2341 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     2401 ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     2461 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2521 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     2581 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggca tatggaaggt
     2641 ccggcaggtt acctgcgccg tgcgtaggtt gcgcagctga cccaagaact gggcaccgca
     2701 ttcttccagc aacaacagct gccggcggct atggctgaca cgttcctgga acacctgtgc
     2761 ctgctggata ttgattccca gccagtggcg gctcgcagca cttccattat cgctaccatt
     2821 ggtccagctt cccgttctgt cgatcgcctg aaagaaatga tcaaagcagg catgaacatc
     2881 gctcgtctga acttttctca cggttctcac gaataccacg ctgaaagcat cgcgaatatc
     2941 cgtgaagcta ccgaatcctt cgcgacctcc ccgctgagct accgtccggt agcaatcgcg
     3001 ctggacacca aaggcccgga aattcgtacc ggcgtgctgc agggtggtcc ggaatccgaa
     3061 gttgaaattg tgaaaggttc tcaggtgctg gtgaccgttg acccgaaatt ccaaacgcgt
     3121 ggcgatgcaa aaactgtgtg ggtagactac cacaacatta ctcgtgtggt agcggtaggt
     3181 ggccgtatct acatcgatga tggcctgatc tctctggtgg ttcagaaaat cggtccggaa
     3241 ggtctggtta ccgaagttga gcacggtggt atcctgggtt ctcgtaaagg cgttaacctg
     3301 cctaacacgg aagttgacct gccgggcctg agcgaacagg atctgctgga tctgcgtttc
     3361 ggcgttcaac acaacgtgga tattatcttc gcatccttcg ttcgtaaagc gtctgacgtg
     3421 ctggctgtac gtgatgcgct gggtccagaa ggtcagaata tcaagatcat ctctaaaatt
     3481 gaaaatcacg aaggcgtgaa aaaatttgac gaaatcctgg aagtttccga tggcattatg
     3541 gtggcgcgcg gcgacctggg cattgaaatt ccggccgaga aagttttcct ggctcaaaag
     3601 atgatgatcg gtcgctgtaa cctggctggc aagccggtag tatgcgcgac ccagatgctg
     3661 gagagcatga tcactaaagc ccgcccaact cgcgccgaaa ccagcgatgt tgcaaacgct
     3721 gtgctggacg gtgcagattg cattatgctg tctggcgaaa ccgcaaaagg ttcttttccg
     3781 gttgaagctg ttatgatgca acacgcaatc gcgcgtgaag ctgaagcggc cgtgtaccac
     3841 cgccagctgt tcgaagaact gcgccgtgct gcaccgctgt ctcgcgaccc gactgaagtt
     3901 accgctatcg gcgcagttga ggcttccttt aaatgttgcg ccgccgctat tatcgtcctg
     3961 actaaaaccg gccgctccgc ccaactgctg tctcagtatc gcccacgcgc tgctgttatc
     4021 gccgttacgc gcagcgcgca ggcagcccgt caggttcacc tgtctcgtgg cgttttcccg
     4081 ctgctgtacc gtgaaccgcc tgaagcgatc tgggccgacg atgtcgatcg tcgtgttcag
     4141 tttggtattg aatccggcaa actgcgtggt ttcctgcgtg ttggtgacct ggttatcgtc
     4201 gttactggtt ggcgtccggg ttctggttat accaacatta tgcgcgttct gtctgtgtcc
     4261 caccaccatc accaccacta agcttgagct cttggctgtt ttggcggatg agagaagatt
     4321 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct
     4381 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
     4441 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat
     4501 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     4561 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     4621 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
     4681 catcctgacg gatggccttt ttgcgtttct acaaactcct cgagcttgat ccggctgcta
     4741 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     4801 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     4861 gatctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4921 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4981 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     5041 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5101 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     5161 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5221 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     5281 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5341 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     5401 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5461 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5521 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5581 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5641 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5701 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5761 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5821 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5881 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5941 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6001 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6061 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6121 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6181 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6241 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6301 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6361 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6421 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6481 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6541 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6601 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6661 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6721 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6781 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6841 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6901 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6961 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7021 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7081 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7141 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7201 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     7261 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagccaaa
     7321 gggcccaagg cccagt
//
