LOCUS       sequence-205508-        8478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For mammalian expression of Flag-hPKL S113A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8478)
  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 8478)
  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: Flag-hPKL S113A
FEATURES             Location/Qualifiers
     source          1..8478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             416..591
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    654..853
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1054..1470
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1400..1422
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1438..1454
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1526..2545
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             1526..2545
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     enhancer        2983..3362
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3363..3566
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3563..3587
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3672..3681
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3681..3704
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     LTR             5382..5975
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6078..6100)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6186..6326
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6241..6260
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6341..6358)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6512..7100)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6592..6611)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7271..8062,8063..8131))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7894..7913
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8132..8236)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8304..8322
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cttttgaaag accccacccg taggtggcaa gctagcttaa gtaacgccac tttgcaaggc
       61 atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa caaagaaaca
      121 gctgaatacc aaacaggata tctgtggtaa gcggttcctg ccccggctca gggccaagaa
      181 cagatgagac agctgagtga tgggccaaac aggatatctg tggtaagcag ttcctgcccc
      241 ggctcggggc caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagtg
      301 aatcatcaga tgtttccagg gtgccccaag gacctgaaaa tgaccctgta ccttatttga
      361 actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tccgctctcc gagctcaata
      421 aaagagccca caacccctca ctcggcgcgc cagtcttccg atagactgcg tcgcccgggt
      481 acccgtattc ccaataaagc ctcttgctgt ttgcatccga atcgtggtct cgctgttcct
      541 tgggagggtc tcctctgagt gattgactac ccacgacggg ggtctttcat ttgggggctc
      601 gtccgggatt tggagacccc tgcccaggga ccaccgaccc accaccggga ggtaagctgg
      661 ccagcaactt atctgtgtct gtccgattgt ctagtgtcta tgtttgatgt tatgcgcctg
      721 cgtctgtact agttagctaa ctagctctgt atctggcgga cccgtggtgg aactgacgag
      781 ttctgaacac ccggccgcaa ccctgggaga cgtcccaggg actttggggg ccgtttttgt
      841 ggcccgacct gaggaaggga gtcgatgtgg aatccgaccc cgtcaggata tgtggttctg
      901 gtaggagacg agaacctaaa acagttcccg cctccgtctg aatttttgct ttcggtttgg
      961 aaccgaagcc gcgcgtcttg tctgctgcag cgctgcagca tcgttctgtg ttgtctctgt
     1021 ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt
     1081 ttgaccttag gtcactggaa agatgtcgag cggatcgctc acaaccagtc ggtagatgtc
     1141 aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg
     1201 ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca
     1261 cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct
     1321 tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct
     1381 ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt
     1441 tatccagccc tcactccttc tctaggcgcc ggaattccga tctgatagct tgccacaacc
     1501 cgtaccaaag atggatagat ccggaaagcc tgaactcacc gcgacgtctg tcgagaagtt
     1561 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
     1621 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
     1681 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat
     1741 tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg
     1801 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc
     1861 ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
     1921 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
     1981 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
     2041 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
     2101 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
     2161 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
     2221 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
     2281 ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact
     2341 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
     2401 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
     2461 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
     2521 cactcgtccg agggcaaagg aatagagtag atgccgaccg aacaagagct gatttcgaga
     2581 acgcctcagc cagcaactcg cgcgagccta gcaaggcaaa tgcgagagaa cggccttacg
     2641 cttggtggca cagttctcgt ccacagttcg ctaagctcgc tcggctgggt cgcgggaggg
     2701 ccggtcgcag tgattcaggc ccttctggat tgtgttggtc cccagggcac gattgtcatg
     2761 cccacgcact cgggtgatct gactgatccc gcagattgga gatcgccgcc cgtgcctgcc
     2821 gattgggtgc agatctctag agctcgctga tccggccatt agccatatta ttcattggtt
     2881 atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcca tatcataata
     2941 tgtacattta tattggctca tgtccaacat taccgccatg ttgacattga ttattgacta
     3001 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     3061 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     3121 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     3181 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     3241 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     3301 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     3361 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     3421 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     3481 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcatgtac
     3541 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
     3601 atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc cgcggcccca
     3661 agcttgttaa cgccaccatg gactacaaag acgatgacga taaagcagaa gggccagcgg
     3721 ggtatctgcg gcgggccagt gtggcccaac tgacccagga gctgggcact gccttcttcc
     3781 agcagcagca gctgccagct gctatggcag acaccttcct ggaacacctc tgcctactgg
     3841 acattgactc cgagcccgtg gctgctcgca gtaccagcat cattgccacc atcgggccag
     3901 catctcgctc cgtggagcgc ctcaaggaga tgatcaaggc cgggatgaac attgcgcgac
     3961 tcaacttctc ccacggctcc cacgagtacc atgctgagtc catcgccaac gtccgggagg
     4021 cggtggagag ctttgcaggt gccccactca gctaccggcc cgtggccatc gccctggaca
     4081 ccaagggacc ggagatccgc actgggatcc tgcagggggg tccagagtcg gaagtggagc
     4141 tggtgaaggg ctcccaggtg ctggtgactg tggaccccgc gttccggacg cgggggaacg
     4201 cgaacaccgt gtgggtggac taccccaata ttgtccgggt cgtgccggtg gggggccgca
     4261 tctacattga cgacgggctc atctccctag tggtccagaa aatcggccca gagggactgg
     4321 tgacccaagt ggagaacggc ggcgtcctgg gcagccggaa gggcgtgaac ttgccagggg
     4381 cccaggtgga cttgcccggg ctgtccgagc aggacgtccg agacctgcgc ttcggggtgg
     4441 agcatggggt ggacatcgtc tttgcctcct ttgtgcggaa agccagcgac gtggctgccg
     4501 tcagggctgc tctgggtccg gaaggacacg gcatcaagat catcagcaaa attgagaacc
     4561 acgaaggcgt gaagaggttt gatgaaatcc tggaggtgag cgacggcatc atggtggcac
     4621 ggggggacct aggcatcgag atcccagcag agaaggtttt cctggctcag aagatgatga
     4681 ttgggcgctg caacttggcg ggcaagcctg ttgtctgtgc cacacagatg ctggagagca
     4741 tgattaccaa gccccggcca acgagggcag agacaagcga tgtcgccaat gctgtgctgg
     4801 atggggctga ctgcatcatg ctgtcagggg agactgccaa gggcaacttc cctgtggaag
     4861 cggtgaagat gcagcatgcg attgcccggg aggcagaggc cgcagtgtac caccggcagc
     4921 tgtttgagga gctacgtcgg gcagcgccac taagccgtga tcccactgag gtcaccgcca
     4981 ttggtgctgt ggaggctgcc ttcaagtgct gtgctgctgc catcattgtg ctgaccacaa
     5041 ctggccgctc agcccagctt ctgtctcggt accgacctcg ggcagcagtc attgctgtca
     5101 cccgctctgc ccaggctgcc cgccaggtcc acttatgccg aggagtcttc cccttgcttt
     5161 accgtgaacc tccagaagcc atctgggcag atgatgtaga tcgccgggtg caatttggca
     5221 ttgaaagtgg aaagctccgt ggcttcctcc gtgttggaga cctggtgatt gtggtgacag
     5281 gctggcgacc tggctccggc tacaccaaca tcatgagggt gctaagcata tcctgaatcg
     5341 ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt
     5401 aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg
     5461 agaatagaga agttcagatc aaggtcagga acagatggaa cagctgaata tgggccaaac
     5521 aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggaacagct
     5581 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
     5641 acagatggtc cccagatgcg gtccagccct cagcagtttc tagagaacca tcagatgttt
     5701 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
     5761 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
     5821 ctcactcggg gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata
     5881 aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg
     5941 agtgattgac tacccgtcag cgggggtctt tcatttgggg gctcgtccgg gatcgggaga
     6001 cccctgccca gggaccaccg acccaccacc gggaggtaag ctggctgcct cgcgcgtttc
     6061 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     6121 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     6181 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     6241 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
     6301 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     6361 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6421 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6481 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6541 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6601 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6661 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6721 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6781 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6841 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6901 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     6961 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     7021 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     7081 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     7141 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     7201 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7261 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7321 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7381 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7441 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7501 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7561 gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta
     7621 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7681 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7741 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7801 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7861 gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt
     7921 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7981 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     8041 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8101 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     8161 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8221 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     8281 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat
     8341 tcataccaga tcaccgaaaa ctgtcctcca aatgtgtccc cctcacactc ccaaattcgc
     8401 gggcttctgc ctcttagacc actctaccct attccccaca ctcaccggag ccaaagccgc
     8461 ggcccttccg tttctttg
//
