LOCUS       sequence-313221-        7810 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For mammalian expression of WT AMPK alpha1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7810)
  AUTHORS   Young NP, Kamireddy A, Van Nostrand JL, Eichner LJ, Shokhirev MN, 
            Dayn Y, Shaw RJ
  TITLE     AMPK governs lineage specification through Tfeb-dependent regulation
            of lysosomes.
  JOURNAL   Genes Dev. 2016 Mar 1;30(5):535-52. doi: 10.1101/gad.274142.115.
  PUBMED    26944679
REFERENCE   2  (bases 1 to 7810)
  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: 
            "10.1101/gad.274142"; journalName: "Genes Dev"; date: "2016-03-1- 
            1"; volume: "30"; issue: "5"; pages: "535-52"
COMMENT     Sequence Label: pCIP-AMPKa1_WT
FEATURES             Location/Qualifiers
     source          1..7810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        78..457
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          691..1707
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1631..1653
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1715..1734
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     misc_feature    join(3488..4062,4063..4065,4066..4074)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3655..3672)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3882..3901
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4075..4674
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4075..4094)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4571..4591
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4755..4772)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4761..4968
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5022..5042)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5022..5040)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5061..5077)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5061..5077)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5074..5096)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5085..5101
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5109..5115,5116..5133,5134..5139))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5154..5175
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5292..5309)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5463..6051)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5543..5562)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6222..7013,7014..7082))
                     /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     6845..6864
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7083..7187)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7214..7670
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7301..7320)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7511..7532
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(7796..7810,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(7810,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg
       61 ggccccccct cgaggtcgac attgattatt gactagttat taatagtaat caattacggg
      121 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      181 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      241 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      301 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      361 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      421 gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg
      481 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
      541 attattttgt gcagcgatgg gggcgggggg gggggggcga ggcggagagg tgcggcggca
      601 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg
      661 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg
      721 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
      781 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga
      841 cggcttgttt cttttctgtg gctgcgtgaa agccttgagg ggctccggga gggccctttg
      901 tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc
      961 ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc
     1021 cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg ggggggctgc
     1081 gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc
     1141 gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc gagttgctga gcacggcccg
     1201 gcttcgggtg cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg
     1261 tggcggcagg tgggggtgcc gggcggggcg gggccgcctc gggccgggga gggctcgggg
     1321 gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat
     1381 tgccttttat ggtaatcgtg cgagagggcg cagggacttc ctttgtccca aatctgtgcg
     1441 gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc
     1501 ggcgccggca ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc
     1561 cttctccctc tccagcctcg gggctgtccg cggggggacg gctgccttcg ggggggacgg
     1621 ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc tgctaaccat
     1681 gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttat tgtgctgtct
     1741 catcattttg gcaaagaatt caacgcggct agcgccatgg cgacagccga gaagcagaaa
     1801 cacgacgggc gggtgaagat cggccactac attctgggtg acacgctggg ggtcggcacc
     1861 ttcggcaaag tgaaggttgg caaacatgaa ttgactgggc ataaagtagc tgtgaagata
     1921 ctcaatcgac agaagattcg gagccttgat gtggtaggaa aaatccgcag agaaattcag
     1981 aacctcaagc ttttcaggca tcctcatata attaaactgt accaggtcat cagtacacca
     2041 tctgatattt tcatggtgat ggaatatgtc tcaggaggag agctatttga ttatatctgt
     2101 aagaatggaa ggctggatga aaaagaaagt cggcgtctgt tccaacagat cctttctggt
     2161 gtggattatt gtcacaggca tatggtggtc catagagatt tgaaacctga aaatgtcctg
     2221 cttgatgcac acatgaatgc aaagatagct gattttggtc tttcaaacat gatgtcagat
     2281 ggtgaatttt taagaacaag ttgtggctca cccaactatg ctgcaccaga agtaatttca
     2341 ggaagattgt atgcaggccc agaggtagat atatggagca gtggggttat tctctatgct
     2401 ttattatgtg gaacccttcc atttgatgat gaccatgtgc caactctttt taagaagata
     2461 tgtgatggga tcttctatac ccctcaatat ttaaatcctt ctgtgattag ccttttgaaa
     2521 catatgctgc aggtggatcc catgaagagg gccacaatca aagatatcag ggaacatgaa
     2581 tggtttaaac aggaccttcc aaaatatctc tttcctgagg atccatcata tagttcaacc
     2641 atgattgatg atgaagcctt aaaagaagta tgtgaaaagt ttgagtgctc agaagaggaa
     2701 gttctcagct gtctttacaa cagaaatcac caggatcctt tggcagttgc ctaccatctc
     2761 ataatagata acaggagaat aatgaatgaa gccaaagatt tctatttggc gacaagccca
     2821 cctgattctt ttcttgatga tcatcacctg actcggcccc atcctgaaag agtaccattc
     2881 ttggttgctg aaacaccaag ggcacgccat acccttgatg aattaaatcc acagaaatcc
     2941 aaacaccaag gtgtaaggaa agcaaaatgg catttaggaa ttagaagtca aagtcgacca
     3001 aatgatatta tggcagaagt atgtagagca atcaaacaat tggattatga atggaaggtt
     3061 gtaaacccat attatttgcg tgtacgaagg aagaatcctg tgacaagcac ttactccaaa
     3121 atgagtctac agttatacca agtggatagt agaacttatc tactggattt ccgtagtatt
     3181 gatgatgaaa ttacagaagc caaatcaggg actgctactc cacagagatc gggatcagtt
     3241 agcaactatc gatcttgcca aaggagtgat tcagatgctg aggctcaagg aaaatcctca
     3301 gaagtttctc ttacctcatc tgtgacctca cttgactctt ctcctgttga cctaactcca
     3361 agacctggaa gtcacacaat agaatttttt gagatgtgtg caaatctaat taaaattctt
     3421 gcacaataag ttaactatgg caattggatc cgatctagat cgagttaatt aagaattccg
     3481 cccccccccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag
     3541 gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga
     3601 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg
     3661 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt
     3721 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca
     3781 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc
     3841 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat
     3901 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc
     3961 ctcggtacac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga
     4021 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatgacc
     4081 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     4141 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     4201 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     4261 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     4321 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     4381 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     4441 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     4501 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     4561 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     4621 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg
     4681 ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc gatgatctag
     4741 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     4801 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     4861 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     4921 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     4981 tatctgcggc cgccaccgcg gtggagctcc agcttttgtt ccctttagtg agggttaatt
     5041 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5101 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5161 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5221 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5281 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5341 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5401 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5461 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5521 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5581 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5641 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5701 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5761 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5821 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5881 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5941 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6001 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6061 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6121 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6181 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6241 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6301 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6361 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6421 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6481 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6541 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6601 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6661 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6721 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6781 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6841 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6901 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6961 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7021 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7081 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7141 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7201 aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     7261 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     7321 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     7381 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     7441 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     7501 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     7561 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa atgagctgat
     7621 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt ccattcgcca
     7681 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     7741 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     7801 tcacgacgtt
//
