LOCUS       sequence-205510-        8478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For mammalian expression of Flag-hPKL S113D.
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 S113D
FEATURES             Location/Qualifiers
     source          1..8478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             426..601
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    664..863
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1064..1480
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1410..1432
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1448..1464
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1536..2555
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             1536..2555
                     /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        2993..3372
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3373..3576
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3573..3597
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3682..3691
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3691..3714
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     LTR             5392..5985
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6088..6110)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6196..6336
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6251..6270
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6351..6368)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6522..7110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6602..6621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7281..8072,8073..8141))
                     /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     7904..7923
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8142..8246)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8314..8332
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgtttctttg cttttgaaag accccacccg taggtggcaa gctagcttaa gtaacgccac
       61 tttgcaaggc atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa
      121 caaagaaaca gctgaatacc aaacaggata tctgtggtaa gcggttcctg ccccggctca
      181 gggccaagaa cagatgagac agctgagtga tgggccaaac aggatatctg tggtaagcag
      241 ttcctgcccc ggctcggggc caagaacaga tggtccccag atgcggtcca gccctcagca
      301 gtttctagtg aatcatcaga tgtttccagg gtgccccaag gacctgaaaa tgaccctgta
      361 ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tccgctctcc
      421 gagctcaata aaagagccca caacccctca ctcggcgcgc cagtcttccg atagactgcg
      481 tcgcccgggt acccgtattc ccaataaagc ctcttgctgt ttgcatccga atcgtggtct
      541 cgctgttcct tgggagggtc tcctctgagt gattgactac ccacgacggg ggtctttcat
      601 ttgggggctc gtccgggatt tggagacccc tgcccaggga ccaccgaccc accaccggga
      661 ggtaagctgg ccagcaactt atctgtgtct gtccgattgt ctagtgtcta tgtttgatgt
      721 tatgcgcctg cgtctgtact agttagctaa ctagctctgt atctggcgga cccgtggtgg
      781 aactgacgag ttctgaacac ccggccgcaa ccctgggaga cgtcccaggg actttggggg
      841 ccgtttttgt ggcccgacct gaggaaggga gtcgatgtgg aatccgaccc cgtcaggata
      901 tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg aatttttgct
      961 ttcggtttgg aaccgaagcc gcgcgtcttg tctgctgcag cgctgcagca tcgttctgtg
     1021 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac
     1081 tcccttaagt ttgaccttag gtcactggaa agatgtcgag cggatcgctc acaaccagtc
     1141 ggtagatgtc aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa
     1201 cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa
     1261 ggtcttttca cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga
     1321 agccttggct tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc
     1381 tcctcttcct ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg
     1441 atcctccctt tatccagccc tcactccttc tctaggcgcc ggaattccga tctgatagct
     1501 tgccacaacc cgtaccaaag atggatagat ccggaaagcc tgaactcacc gcgacgtctg
     1561 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     1621 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     1681 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     1741 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     1801 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     1861 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     1921 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     1981 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     2041 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     2101 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     2161 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     2221 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     2281 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     2341 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     2401 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     2461 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     2521 gacgccccag cactcgtccg agggcaaagg aatagagtag atgccgaccg aacaagagct
     2581 gatttcgaga acgcctcagc cagcaactcg cgcgagccta gcaaggcaaa tgcgagagaa
     2641 cggccttacg cttggtggca cagttctcgt ccacagttcg ctaagctcgc tcggctgggt
     2701 cgcgggaggg ccggtcgcag tgattcaggc ccttctggat tgtgttggtc cccagggcac
     2761 gattgtcatg cccacgcact cgggtgatct gactgatccc gcagattgga gatcgccgcc
     2821 cgtgcctgcc gattgggtgc agatctctag agctcgctga tccggccatt agccatatta
     2881 ttcattggtt atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcca
     2941 tatcataata tgtacattta tattggctca tgtccaacat taccgccatg ttgacattga
     3001 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     3061 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     3121 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     3181 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     3241 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     3301 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     3361 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     3421 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     3481 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     3541 aggcatgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagatcgcc
     3601 tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc
     3661 cgcggcccca agcttgttaa cgccaccatg gactacaaag acgatgacga taaagcagaa
     3721 gggccagcgg ggtatctgcg gcgggccagt gtggcccaac tgacccagga gctgggcact
     3781 gccttcttcc agcagcagca gctgccagct gctatggcag acaccttcct ggaacacctc
     3841 tgcctactgg acattgactc cgagcccgtg gctgctcgca gtaccagcat cattgccacc
     3901 atcgggccag catctcgctc cgtggagcgc ctcaaggaga tgatcaaggc cgggatgaac
     3961 attgcgcgac tcaacttctc ccacggctcc cacgagtacc atgctgagtc catcgccaac
     4021 gtccgggagg cggtggagag ctttgcaggt gacccactca gctaccggcc cgtggccatc
     4081 gccctggaca ccaagggacc ggagatccgc actgggatcc tgcagggggg tccagagtcg
     4141 gaagtggagc tggtgaaggg ctcccaggtg ctggtgactg tggaccccgc gttccggacg
     4201 cgggggaacg cgaacaccgt gtgggtggac taccccaata ttgtccgggt cgtgccggtg
     4261 gggggccgca tctacattga cgacgggctc atctccctag tggtccagaa aatcggccca
     4321 gagggactgg tgacccaagt ggagaacggc ggcgtcctgg gcagccggaa gggcgtgaac
     4381 ttgccagggg cccaggtgga cttgcccggg ctgtccgagc aggacgtccg agacctgcgc
     4441 ttcggggtgg agcatggggt ggacatcgtc tttgcctcct ttgtgcggaa agccagcgac
     4501 gtggctgccg tcagggctgc tctgggtccg gaaggacacg gcatcaagat catcagcaaa
     4561 attgagaacc acgaaggcgt gaagaggttt gatgaaatcc tggaggtgag cgacggcatc
     4621 atggtggcac ggggggacct aggcatcgag atcccagcag agaaggtttt cctggctcag
     4681 aagatgatga ttgggcgctg caacttggcg ggcaagcctg ttgtctgtgc cacacagatg
     4741 ctggagagca tgattaccaa gccccggcca acgagggcag agacaagcga tgtcgccaat
     4801 gctgtgctgg atggggctga ctgcatcatg ctgtcagggg agactgccaa gggcaacttc
     4861 cctgtggaag cggtgaagat gcagcatgcg attgcccggg aggcagaggc cgcagtgtac
     4921 caccggcagc tgtttgagga gctacgtcgg gcagcgccac taagccgtga tcccactgag
     4981 gtcaccgcca ttggtgctgt ggaggctgcc ttcaagtgct gtgctgctgc catcattgtg
     5041 ctgaccacaa ctggccgctc agcccagctt ctgtctcggt accgacctcg ggcagcagtc
     5101 attgctgtca cccgctctgc ccaggctgcc cgccaggtcc acttatgccg aggagtcttc
     5161 cccttgcttt accgtgaacc tccagaagcc atctgggcag atgatgtaga tcgccgggtg
     5221 caatttggca ttgaaagtgg aaagctccgt ggcttcctcc gtgttggaga cctggtgatt
     5281 gtggtgacag gctggcgacc tggctccggc tacaccaaca tcatgagggt gctaagcata
     5341 tcctgaatcg ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga
     5401 ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaaa
     5461 tacataactg agaatagaga agttcagatc aaggtcagga acagatggaa cagctgaata
     5521 tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga
     5581 tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc
     5641 agggccaaga acagatggtc cccagatgcg gtccagccct cagcagtttc tagagaacca
     5701 tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac
     5761 caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag
     5821 cccacaaccc ctcactcggg gcgccagtcc tccgattgac tgagtcgccc gggtacccgt
     5881 gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg
     5941 gtctcctctg agtgattgac tacccgtcag cgggggtctt tcatttgggg gctcgtccgg
     6001 gatcgggaga cccctgccca gggaccaccg acccaccacc gggaggtaag ctggctgcct
     6061 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     6121 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     6181 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     6241 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     6301 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     6361 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6421 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6481 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6541 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6601 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6661 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6721 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6781 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6841 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6901 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6961 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     7021 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     7081 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     7141 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7201 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7261 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7321 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7381 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7441 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7501 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7561 ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg
     7621 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7681 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7741 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7801 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7861 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat
     7921 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7981 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     8041 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     8101 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     8161 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8221 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     8281 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     8341 cttcaagaat tcataccaga tcaccgaaaa ctgtcctcca aatgtgtccc cctcacactc
     8401 ccaaattcgc gggcttctgc ctcttagacc actctaccct attccccaca ctcaccggag
     8461 ccaaagccgc ggcccttc
//
