LOCUS       sequence-202151-        8477 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 8477)
  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 8477)
  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..8477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             414..589
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    652..851
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1052..1468
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1398..1420
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1436..1452
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1525..2544
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             1525..2544
                     /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        2980..3359
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3360..3563
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3560..3584
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3669..3678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3678..3701
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     LTR             5379..5972
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6075..6097)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6183..6323
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6238..6257
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6338..6355)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6509..7097)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6589..6608)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7268..8059,8060..8128))
                     /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     7891..7910
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8129..8233)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8301..8319
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tttgaaagac cccacccgta ggtggcaagc tagcttaagt aacgccactt tgcaaggcat
       61 ggaaaaatac ataactgaga atagaaaagt tcagatcaag gtcaggaaca aagaaacagc
      121 tgaataccaa acaggatatc tgtggtaagc ggttcctgcc ccggctcagg gccaagaaca
      181 gatgagacag ctgagtgatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg
      241 ctcggggcca agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagtgaa
      301 tcatcagatg tttccagggt gccccaagga cctgaaaatg accctgtacc ttatttgaac
      361 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc cgctctccga gctcaataaa
      421 agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc gcccgggtac
      481 ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg ctgttccttg
      541 ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt gggggctcgt
      601 ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg taagctggcc
      661 agcaacttat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg
      721 tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt
      781 ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg
      841 cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt
      901 aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa
      961 ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct
     1021 gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt
     1081 gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa
     1141 gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc
     1201 gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc
     1261 tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt
     1321 tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc
     1381 atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta
     1441 tccagccctc actccttctc taggcgccgg aattccgatc tgatcagctt gccacaaccc
     1501 gtaccaaaga tggatagatc cggaaagcct gaactcaccg cgacgtctgt cgagaagttt
     1561 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     1621 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     1681 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     1741 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     1801 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     1861 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     1921 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     1981 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     2041 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     2101 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     2161 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     2221 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     2281 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     2341 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     2401 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     2461 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     2521 actcgtccga gggcaaagga atagagtaga tgccgaccga acaagagctg atttcgagaa
     2581 cgcctcagcc agcaactcgc gcgagcctag caaggcaaat gcgagagaac ggccttacgc
     2641 ttggtggcac agttctcgtc cacagttcgc taagctcgct cggctgggtc gcgggagggc
     2701 cggtcgcagt gattcaggcc cttctggatt gtgttggtcc ccagggcacg attgtcatgc
     2761 ccacgcactc gggtgatctg actgatcccg cagattggag atcgccgccc gtgcctgccg
     2821 attgggtgca gatctagagc tcgctgatcc ggccattagc catattattc attggttata
     2881 tagcataaat caatattggc tattggccat tgcatacgtt gtatccatat cataatatgt
     2941 acatttatat tggctcatgt ccaacattac cgccatgttg acattgatta ttgactagtt
     3001 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     3061 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     3121 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     3181 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     3241 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     3301 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     3361 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     3421 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     3481 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg catgtacggt
     3541 gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc
     3601 cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgc ggccccaagc
     3661 ttgttaacgc caccatggac tacaaagacg atgacgataa agcagaaggg ccagcggggt
     3721 atctgcggcg ggccagtgtg gcccaactga cccaggagct gggcactgcc ttcttccagc
     3781 agcagcagct gccagctgct atggcagaca ccttcctgga acacctctgc ctactggaca
     3841 ttgactccga gcccgtggct gctcgcagta ccagcatcat tgccaccatc gggccagcat
     3901 ctcgctccgt ggagcgcctc aaggagatga tcaaggccgg gatgaacatt gcgcgactca
     3961 acttctccca cggctcccac gagtaccatg ctgagtccat cgccaacgtc cgggaggcgg
     4021 tggagagctt tgcaggtgcc ccactcagct accggcccgt ggccatcgcc ctggacacca
     4081 agggaccgga gatccgcact gggatcctgc aggggggtcc agagtcggaa gtggagctgg
     4141 tgaagggctc ccaggtgctg gtgactgtgg accccgcgtt ccggacgcgg gggaacgcga
     4201 acaccgtgtg ggtggactac cccaatattg tccgggtcgt gccggtgggg ggccgcatct
     4261 acattgacga cgggctcatc tccctagtgg tccagaaaat cggcccagag ggactggtga
     4321 cccaagtgga gaacggcggc gtcctgggca gccggaaggg cgtgaacttg ccaggggccc
     4381 aggtggactt gcccgggctg tccgagcagg acgtccgaga cctgcgcttc ggggtggagc
     4441 atggggtgga catcgtcttt gcctcctttg tgcggaaagc cagcgacgtg gctgccgtca
     4501 gggctgctct gggtccggaa ggacacggca tcaagatcat cagcaaaatt gagaaccacg
     4561 aaggcgtgaa gaggtttgat gaaatcctgg aggtgagcga cggcatcatg gtggcacggg
     4621 gggacctagg catcgagatc ccagcagaga aggttttcct ggctcagaag atgatgattg
     4681 ggcgctgcaa cttggcgggc aagcctgttg tctgtgccac acagatgctg gagagcatga
     4741 ttaccaagcc ccggccaacg agggcagaga caagcgatgt cgccaatgct gtgctggatg
     4801 gggctgactg catcatgctg tcaggggaga ctgccaaggg caacttccct gtggaagcgg
     4861 tgaagatgca gcatgcgatt gcccgggagg cagaggccgc agtgtaccac cggcagctgt
     4921 ttgaggagct acgtcgggca gcgccactaa gccgtgatcc cactgaggtc accgccattg
     4981 gtgctgtgga ggctgccttc aagtgctgtg ctgctgccat cattgtgctg accacaactg
     5041 gccgctcagc ccagcttctg tctcggtacc gacctcgggc agcagtcatt gctgtcaccc
     5101 gctctgccca ggctgcccgc caggtccact tatgccgagg agtcttcccc ttgctttacc
     5161 gtgaacctcc agaagccatc tgggcagatg atgtagatcg ccgggtgcaa tttggcattg
     5221 aaagtggaaa gctccgtggc ttcctccgtg ttggagacct ggtgattgtg gtgacaggct
     5281 ggcgacctgg ctccggctac accaacatca tgagggtgct aagcatatcc tgaatcgata
     5341 aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg
     5401 tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaaatac ataactgaga
     5461 atagagaagt tcagatcaag gtcaggaaca gatggaacag ctgaatatgg gccaaacagg
     5521 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg aacagctgaa
     5581 tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca
     5641 gatggtcccc agatgcggtc cagccctcag cagtttctag agaaccatca gatgtttcca
     5701 gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct
     5761 tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc
     5821 actcggggcg ccagtcctcc gattgactga gtcgcccggg tacccgtgta tccaataaac
     5881 cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt
     5941 gattgactac ccgtcagcgg gggtctttca tttgggggct cgtccgggat cgggagaccc
     6001 ctgcccaggg accaccgacc caccaccggg aggtaagctg gctgcctcgc gcgtttcggt
     6061 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     6121 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     6181 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
     6241 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
     6301 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6361 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6421 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6481 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6541 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6601 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6661 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6721 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6781 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6841 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6901 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     6961 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     7021 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     7081 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     7141 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     7201 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     7261 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     7321 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7381 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7441 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7501 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7561 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7621 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7681 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7741 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7801 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7861 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa
     7921 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7981 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     8041 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     8101 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     8161 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     8221 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     8281 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattca
     8341 taccagatca ccgaaaactg tcctccaaat gtgtccccct cacactccca aattcgcggg
     8401 cttctgcctc ttagaccact ctaccctatt ccccacactc accggagcca aagccgcggc
     8461 ccttccgttt ctttgct
//
