LOCUS       sequence-458387-        7135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of an shRNA construct that knocks down rat MPC1. This 
            plasmid also encodes a blue fluorescent protein tag to verify 
            transfection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7135)
  AUTHORS   Tiwari A, Myeong J, Hashemiaghdam A, Stunault MI, Zhang H, Niu X, 
            Laramie MA, Sponagel J, Shriver LP, Patti GJ, Klyachko VA, Ashrafi G
  TITLE     Mitochondrial pyruvate transport regulates presynaptic metabolism 
            and neurotransmission.
  JOURNAL   Sci Adv. 2024 Nov 15;10(46):eadp7423. doi: 10.1126/sciadv.adp7423. 
            Epub 2024 Nov 15.
  PUBMED    39546604
REFERENCE   2  (bases 1 to 7135)
  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: "Sci Adv."; 
            date: "2024-11-15"; volume: "10(46):eadp7423. doi"; pages: " 
            10.1126/sciadv.adp7423. Epub 2024 Nov 15"
COMMENT     Sequence Label: pLKO.1-mTagBFP2-rMPC1 shRNA
FEATURES             Location/Qualifiers
     source          1..7135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     77..94
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    211..232
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(247..252,253..270,271..277)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    285..301
                     /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)."
     primer_bind     290..312
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     309..325
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     309..325
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     344..364
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        346..364
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        392..618
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             619..799
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    846..971
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1464..1697
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1882..1926
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        2224..2472
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2224..2244
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2395..2414
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2569..2686
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2735..3245
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3164..3182
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      3254..3263
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3260..3970
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     LTR             4045..4278
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4350..4471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4387..4406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4441..4460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4511..4646
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4666..4685)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4667..4685)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4695..4712)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4695..4711)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4704..4726)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4853..5308
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4940..4959)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5150..5171
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5334..5438
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5439..5507,5508..6299)
                     /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(5657..5676)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6470..7058
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6959..6978
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
       61 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
      121 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
      181 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
      241 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
      301 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
      361 aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct
      421 tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca
      481 tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt
      541 ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc
      601 ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
      661 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
      721 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
      781 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
      841 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
      901 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
      961 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     1021 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     1081 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     1141 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     1201 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     1261 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc
     1321 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     1381 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     1441 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     1501 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     1561 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     1621 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     1681 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     1741 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     1801 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     1861 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     1921 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     1981 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     2041 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc
     2101 gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag atccattcga
     2161 ttagtgaacg gatctcgacg gtatcgatca cgagactagc ctcgagcggc cgcccccttc
     2221 accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     2281 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     2341 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     2401 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     2461 ggacgaaaca ccggcaaacg aagtcgctca gctcactcga gtgagctgag cgacttcgtt
     2521 tgtttttgaa ttctcgacct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     2581 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     2641 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     2701 ggacagcaga gatccacttt ggccgcggct cgagggggtt ggggttgcgc cttttccaag
     2761 gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg
     2821 gcgccgaccc tgggtctcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg
     2881 ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt
     2941 ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc actagtaccc
     3001 tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca
     3061 atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg
     3121 cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca
     3181 agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc
     3241 cccaggggga tccgccacca tggtgtctaa gggcgaagag ctgattaagg agaacatgca
     3301 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg
     3361 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
     3421 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat
     3481 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg
     3541 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     3601 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg
     3661 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc tgtaccccgc
     3721 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct
     3781 gatcgcaaac gccaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc
     3841 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgagaccta
     3901 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca
     3961 caagcttaat taaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     4021 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc
     4081 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     4141 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     4201 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     4261 tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg
     4321 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     4381 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4441 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     4501 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4561 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     4621 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     4681 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     4741 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     4801 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     4861 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4921 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4981 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5041 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     5101 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5161 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     5221 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5281 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     5341 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5401 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5461 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5521 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5581 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5641 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5701 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5761 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5821 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5881 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5941 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6001 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6061 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6121 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6181 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6241 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6301 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6361 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6421 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6481 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6541 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6601 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6661 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6721 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6781 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6841 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6901 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6961 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7021 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7081 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatc
//
