LOCUS       sequence-205506-        8478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For mammalian expression of Flag-hPKL WT.
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 WT
FEATURES             Location/Qualifiers
     source          1..8478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             419..594
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    657..856
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1057..1473
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1403..1425
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1441..1457
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1529..2548
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             1529..2548
                     /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        2986..3365
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3366..3569
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3566..3590
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3675..3684
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3684..3707
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     LTR             5385..5978
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6081..6103)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6189..6329
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6244..6263
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6344..6361)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6515..7103)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6595..6614)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7274..8065,8066..8134))
                     /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     7897..7916
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8135..8239)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8307..8325
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttgcttttga aagaccccac ccgtaggtgg caagctagct taagtaacgc cactttgcaa
       61 ggcatggaaa aatacataac tgagaataga aaagttcaga tcaaggtcag gaacaaagaa
      121 acagctgaat accaaacagg atatctgtgg taagcggttc ctgccccggc tcagggccaa
      181 gaacagatga gacagctgag tgatgggcca aacaggatat ctgtggtaag cagttcctgc
      241 cccggctcgg ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta
      301 gtgaatcatc agatgtttcc agggtgcccc aaggacctga aaatgaccct gtaccttatt
      361 tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttccgctc tccgagctca
      421 ataaaagagc ccacaacccc tcactcggcg cgccagtctt ccgatagact gcgtcgcccg
      481 ggtacccgta ttcccaataa agcctcttgc tgtttgcatc cgaatcgtgg tctcgctgtt
      541 ccttgggagg gtctcctctg agtgattgac tacccacgac gggggtcttt catttggggg
      601 ctcgtccggg atttggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc
      661 tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgtttga tgttatgcgc
      721 ctgcgtctgt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac
      781 gagttctgaa cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt
      841 tgtggcccga cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt
      901 ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt
      961 tggaaccgaa gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc
     1021 tgtctgactg tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta
     1081 agtttgacct taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat
     1141 gtcaagaaga gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga
     1201 tggccgcgag acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt
     1261 tcacctggcc cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg
     1321 gcttttgacc cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt
     1381 cctccatccg ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc
     1441 ctttatccag ccctcactcc ttctctaggc gccggaattc cgatctgata gcttgccaca
     1501 acccgtacca aagatggata gatccggaaa gcctgaactc accgcgacgt ctgtcgagaa
     1561 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga
     1621 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg
     1681 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc
     1741 gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg
     1801 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca
     1861 gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt
     1921 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc
     1981 gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc
     2041 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca
     2101 cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc
     2161 ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt
     2221 cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca
     2281 tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca
     2341 actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg
     2401 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag
     2461 cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc
     2521 cagcactcgt ccgagggcaa aggaatagag tagatgccga ccgaacaaga gctgatttcg
     2581 agaacgcctc agccagcaac tcgcgcgagc ctagcaaggc aaatgcgaga gaacggcctt
     2641 acgcttggtg gcacagttct cgtccacagt tcgctaagct cgctcggctg ggtcgcggga
     2701 gggccggtcg cagtgattca ggcccttctg gattgtgttg gtccccaggg cacgattgtc
     2761 atgcccacgc actcgggtga tctgactgat cccgcagatt ggagatcgcc gcccgtgcct
     2821 gccgattggg tgcagatctc tagagctcgc tgatccggcc attagccata ttattcattg
     2881 gttatatagc ataaatcaat attggctatt ggccattgca tacgttgtat ccatatcata
     2941 atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat tgattattga
     3001 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     3061 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     3121 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     3181 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     3241 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     3301 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     3361 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     3421 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     3481 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcatg
     3541 tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac
     3601 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggcc
     3661 ccaagcttgt taacgccacc atggactaca aagacgatga cgataaagca gaagggccag
     3721 cggggtatct gcggcgggcc agtgtggccc aactgaccca ggagctgggc actgccttct
     3781 tccagcagca gcagctgcca gctgctatgg cagacacctt cctggaacac ctctgcctac
     3841 tggacattga ctccgagccc gtggctgctc gcagtaccag catcattgcc accatcgggc
     3901 cagcatctcg ctccgtggag cgcctcaagg agatgatcaa ggccgggatg aacattgcgc
     3961 gactcaactt ctcccacggc tcccacgagt accatgctga gtccatcgcc aacgtccggg
     4021 aggcggtgga gagctttgca ggttccccac tcagctaccg gcccgtggcc atcgccctgg
     4081 acaccaaggg accggagatc cgcactggga tcctgcaggg gggtccagag tcggaagtgg
     4141 agctggtgaa gggctcccag gtgctggtga ctgtggaccc cgcgttccgg acgcggggga
     4201 acgcgaacac cgtgtgggtg gactacccca atattgtccg ggtcgtgccg gtggggggcc
     4261 gcatctacat tgacgacggg ctcatctccc tagtggtcca gaaaatcggc ccagagggac
     4321 tggtgaccca agtggagaac ggcggcgtcc tgggcagccg gaagggcgtg aacttgccag
     4381 gggcccaggt ggacttgccc gggctgtccg agcaggacgt ccgagacctg cgcttcgggg
     4441 tggagcatgg ggtggacatc gtctttgcct cctttgtgcg gaaagccagc gacgtggctg
     4501 ccgtcagggc tgctctgggt ccggaaggac acggcatcaa gatcatcagc aaaattgaga
     4561 accacgaagg cgtgaagagg tttgatgaaa tcctggaggt gagcgacggc atcatggtgg
     4621 cacgggggga cctaggcatc gagatcccag cagagaaggt tttcctggct cagaagatga
     4681 tgattgggcg ctgcaacttg gcgggcaagc ctgttgtctg tgccacacag atgctggaga
     4741 gcatgattac caagccccgg ccaacgaggg cagagacaag cgatgtcgcc aatgctgtgc
     4801 tggatggggc tgactgcatc atgctgtcag gggagactgc caagggcaac ttccctgtgg
     4861 aagcggtgaa gatgcagcat gcgattgccc gggaggcaga ggccgcagtg taccaccggc
     4921 agctgtttga ggagctacgt cgggcagcgc cactaagccg tgatcccact gaggtcaccg
     4981 ccattggtgc tgtggaggct gccttcaagt gctgtgctgc tgccatcatt gtgctgacca
     5041 caactggccg ctcagcccag cttctgtctc ggtaccgacc tcgggcagca gtcattgctg
     5101 tcacccgctc tgcccaggct gcccgccagg tccacttatg ccgaggagtc ttccccttgc
     5161 tttaccgtga acctccagaa gccatctggg cagatgatgt agatcgccgg gtgcaatttg
     5221 gcattgaaag tggaaagctc cgtggcttcc tccgtgttgg agacctggtg attgtggtga
     5281 caggctggcg acctggctcc ggctacacca acatcatgag ggtgctaagc atatcctgaa
     5341 tcgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc
     5401 tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa
     5461 ctgagaatag agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca
     5521 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca
     5581 gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca
     5641 agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg
     5701 tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag
     5761 ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa
     5821 cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca
     5881 ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct
     5941 ctgagtgatt gactacccgt cagcgggggt ctttcatttg ggggctcgtc cgggatcggg
     6001 agacccctgc ccagggacca ccgacccacc accgggaggt aagctggctg cctcgcgcgt
     6061 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     6121 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     6181 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     6241 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     6301 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     6361 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6421 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6481 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6541 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6601 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6661 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6721 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6781 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6841 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6901 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     6961 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     7021 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     7081 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     7141 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     7201 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     7261 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     7321 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     7381 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7441 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7501 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7561 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg
     7621 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7681 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7741 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7801 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7861 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa
     7921 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7981 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     8041 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     8101 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     8161 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     8221 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     8281 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag
     8341 aattcatacc agatcaccga aaactgtcct ccaaatgtgt ccccctcaca ctcccaaatt
     8401 cgcgggcttc tgcctcttag accactctac cctattcccc acactcaccg gagccaaagc
     8461 cgcggccctt ccgtttct
//
