LOCUS       sequence-159337-        8072 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cre-dependent DN-AMPK under EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8072)
  AUTHORS   Kong D, Dagon Y, Campbell JN, Guo Y, Yang Z, Yi X, Aryal P, 
            Wellenstein K, Kahn BB, Sabatini BL, Lowell BB
  TITLE     A Postsynaptic AMPK-->p21-Activated Kinase Pathway Drives 
            Fasting-Induced Synaptic Plasticity in AgRP Neurons.
  JOURNAL   Neuron. 2016 Jun 16. pii: S0896-6273(16)30204-5. doi: 
            10.1016/j.neuron.2016.05.025.
  PUBMED    27321921
REFERENCE   2  (bases 1 to 8072)
  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"; journalName: "Neuron. 
            2016 Jun 16. pii: S0896-6273(16)30204-5. doi: 
            10.1016/j.neuron.2016.05.025."
COMMENT     Sequence Label: pAAV-EF1a-Flex-DN-AMPK-K45R-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..8072
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1450..1483)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1534..1567)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1576..2283)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1854..1873)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2123..2141
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2290..2346)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(4015..4044)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     protein_bind    4057..4090
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    4141..4174
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4199..4787
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4252..4272)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4670..4681)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4819..5295
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5018..5037)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5335..5475
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5335..5464
                     /label=AAV2 ITR
     rep_origin      5550..6005
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5637..5656)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5847..5868
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6022..6041)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6141..6163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6201..6219)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6287..6391
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6392..6460,6461..7252)
                     /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     complement(6610..6629)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7423..8011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7912..7931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgccttact tgtacagctc gtccatgccg ccggtggagt ggcggccctc
     1621 ggcgcgttcg tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt
     1681 gatgttgacg ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt
     1741 gacctcagcg tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt
     1801 cagggcgccg tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt
     1861 ctgcattacg gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa
     1921 ctcgccgtcc tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt
     1981 catcacgcgc tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc
     2041 ggcggggtgc ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca
     2101 ggcgaagggc agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta
     2161 ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat
     2221 gtgcaccttg aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct
     2281 cacggatcta ggtccagggt tctcctccac gtctccagcc tgcttcagca ggctgaagtt
     2341 agtagctccg cttccacggg ctaaagcagt gataagactg gcgcacattt caaaaaaatc
     2401 catggtatga ctgcccaggc gcggggaagc ggaggacaaa gtgctgccag tcaaagaacc
     2461 agtgagagag ccagacagtg aatggttctc ggctgtgctg gaatcgacac ttgaccgagg
     2521 tctgtggagg ccggcagcag aacaggagcg ctgaggtgtt gaagaaccag acctctgctc
     2581 caccacctca tcatcgatgc ttttaaagtc tagaagatag ctccgattgt caaccaggta
     2641 aagctgtaag ctcattttca cgtaattgcc agtcactggg ttttttcttc ttactcgaag
     2701 atggtatgca ttcactacct tccattcaaa gtccagctgc ttcatagctc ggtacacctc
     2761 cgccataatg tcgtatggtt tgctctggct tcggatccca aggtgccact tggctttttt
     2821 cacagctaaa gatttgggct tagttgtgtt gagtgcatcc agtggacagc gtgctttggg
     2881 gctgtctgct atgagaggtg gcatcctttc aggatgtggt ttcaggccgg ggggaatgtg
     2941 catggccata tcgtccatga aggaacccgt tggaggactg gaggcgaggt agaactcact
     3001 ggcttggttc attattctcc gattgtcaat gatgagatga taagccactg cgagctggtc
     3061 ttgagggtca ccactgtata aactgttcat cacttctgat tctgtacact caaatttttc
     3121 acatacttct ttcacagcct catcatcaat gacgttagca tcataggagg ggtcttcagg
     3181 aaagaggtaa ctgggcaaat cctgtttaaa ccattcatgc tctcgtatgt ctttgatagt
     3241 tgctcgcttc aaggggtcca cctgcagcat gtgcatcagc agagtggcaa tagaacggtt
     3301 gagatactcc gggatgtaga acacaccccc tcggatcttc ttaaagagcg taggcacgtg
     3361 ctcatcgtcg aacgggaggg tgccacagag aagggcatac aggataacac cacagctcca
     3421 gatatcaacc tcaggacccg catacagcct tcctgagatg acctccggtg ctgcataatt
     3481 tggcgatcca cagctagttc gtagaaattc accatctgac atcatattag agagtccgaa
     3541 gtcagctatc ttagcattca tctgggcgtc cagcaacacg ttctctggct tcaggtccct
     3601 gtggacaacc atgtgcctgt gacagtagtc cacggcagac agaatctgct ggaagagccg
     3661 gcgagcttcc acctcttcaa ccctcccgtg tttacagatg tagtcgaaca attcacctcc
     3721 agacacatat tccattacca taaaaaagtc tgttggagtg ctgatcactt ggtagagttt
     3781 gataatatga ggatgacgaa agagtttaag attttgaatt tctcgtttta tttttccaac
     3841 aacatctaaa ctgcgaatct tctgtctatt taagatccta actgccactt tatggcctgt
     3901 caattgatgt tctccaatct tcactttgcc gaaggtgccg acgcccaggg tgtcccccag
     3961 cacgtagtgt ccgatcttca cacgcccgtc gtgcttctgc ttctcgccgc caccgaggtc
     4021 ctcctcggag ataagcttct gctccatggc gctagcataa cttcgtataa agtatcctat
     4081 acgaagttat ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga
     4141 ataacttcgt ataatgtatg ctatacgaag ttatgaattc gatatcaagc ttatcgataa
     4201 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     4261 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     4321 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     4381 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     4441 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     4501 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     4561 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     4621 ctatgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     4681 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     4741 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc
     4801 gctgctcgag agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc
     4861 ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt
     4921 ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag
     4981 gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt
     5041 gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg
     5101 cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg
     5161 tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct
     5221 caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct
     5281 cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac
     5341 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     5401 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     5461 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     5521 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     5581 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     5641 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5701 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5761 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5821 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5881 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5941 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     6001 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     6061 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     6121 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     6181 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     6241 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     6301 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     6361 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     6421 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     6481 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     6541 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     6601 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     6661 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     6721 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     6781 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     6841 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     6901 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     6961 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     7021 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     7081 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     7141 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     7201 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     7261 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     7321 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     7381 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     7441 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     7501 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     7561 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     7621 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     7681 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     7741 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     7801 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     7861 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     7921 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7981 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     8041 cctggccttt tgctggcctt ttgctcacat gt
//
