LOCUS       sequence-180162-        4974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses shRNA against TIM23 with a separate promoter for 
            expression of GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4974)
  AUTHORS   Yano H, Baranov SV, Baranova OV, Kim J, Pan Y, Yablonska S, Carlisle
            DL, Ferrante RJ, Kim AH, Friedlander RM
  TITLE     Inhibition of mitochondrial protein import by mutant huntingtin.
  JOURNAL   Nat Neurosci. 2014 Jun;17(6):822-31. doi: 10.1038/nn.3721. Epub 2014
            May 18.
  PUBMED    24836077
REFERENCE   2  (bases 1 to 4974)
  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: "10.1038/nn.3721"; 
            journalName: "Nat Neurosci"; date: "2014-06"; volume: "17"; issue: 
            "6"; pages: "822-31"
COMMENT     Sequence Label: pBSU6-shTim23/CMV-eGFP
FEATURES             Location/Qualifiers
     source          1..4974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(142..933,934..1002))
                     /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     765..784
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1003..1107)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1133..1588)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1270..1291)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1482..1501
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1715..1737
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1729..1746
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1730..1746
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1756..1775
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1756..1774
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1792..1808
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1805..2118
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2037..2057
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     2130..2146
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2131..2147
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2289..2592
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2593..2796
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2747..2767
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    2819..2899
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      2901..2910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2907..2909,2910..2912,2913..3626)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2907..2909,2910..2912,2913..3623)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2952..2973)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3213..3232)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3560..3581
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3749..3830
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3786..3805)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3863..3879)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3916..3936)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3916..3934)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3955..3971)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3955..3971)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3968..3990)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3979..3995
                     /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(4003..4009,4010..4027,4028..4033))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4048..4069
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4186..4203)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4357..4945)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4437..4456)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
       61 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
      121 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
      181 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
      241 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
      301 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
      361 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
      421 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
      481 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
      541 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
      601 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
      661 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
      721 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
      781 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
      841 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
      901 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
      961 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     1021 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1081 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     1141 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     1201 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     1261 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     1321 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     1381 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     1441 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     1501 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     1561 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg
     1621 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     1681 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     1741 gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtacccg ctctagaact
     1801 agtggatccg acgccgccat ctctaggccc gcgccggccc cctcgcacag acttgtggga
     1861 gaagctcggc tactcccctg ccccggttaa tttgcatata atatttccta gtaactatag
     1921 aggcttaatg tgcgataaaa gacagataat ctgttctttt taatactagc tacattttac
     1981 atgataggct tggatttcta taagagatac aaatactaaa ttattatttt aaaaaacagc
     2041 acaaaaggaa actcacccta actgtaaagt aattgtgtgt tttgagacta taaatatccc
     2101 ttggagaaaa gccttgtttg ggccccccct cgaggtcgac ggtatcgcgg aggaagtagc
     2161 aacaaattca agagatttgt tgctacttcc tccgtttttt ggaaatgcag ggtattaccg
     2221 ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     2281 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     2341 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     2401 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2461 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     2521 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2581 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     2641 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     2701 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     2761 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc
     2821 tagcgctacc ggactcagat ctcgagctca agcttcgaat tctgcagtcg acggtaccgc
     2881 gggcccggga tccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     2941 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     3001 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     3061 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     3121 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     3181 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     3241 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     3301 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     3361 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     3421 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3481 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     3541 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3601 tcggcatgga cgagctgtac aagtaaagcg gccgcgactc tagatcataa tcagccatac
     3661 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     3721 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     3781 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3841 tggtttgtcc aaacttccgg gggatccact agttctagag cggccgccac cgcggtggag
     3901 ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
     3961 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4021 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4081 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4141 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4201 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4261 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4321 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4381 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4441 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4501 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4561 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4621 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4681 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4741 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4801 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4861 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4921 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtc
//
