LOCUS       sequence-479483-        6991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Membrane-tethered mCherry shRNA targeting Mfn1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6991)
  AUTHORS   Rodriguez Salazar MP, Kolanukuduru S, Ramirez V, Lyu B, Manigault G,
            Sejourne G, Sesaki H, Yu G, Eroglu C
  TITLE     Mitochondrial fission controls astrocyte morphogenesis and 
            organization in the cortex.
  JOURNAL   J Cell Biol. 2025 Oct 6;224(10):e202410130. doi: 
            10.1083/jcb.202410130. Epub 2025 Sep 3.
  PUBMED    40899994
REFERENCE   2  (bases 1 to 6991)
  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: "J Cell 
            Biol."; date: "2025-10-6"; volume: "224(10):e202410130. doi"; pages:
            " 10.1083/jcb.202410130. Epub 2025 Sep 3"
COMMENT     Sequence Label: pPB-shMfn1-mCherry-CAAX
FEATURES             Location/Qualifiers
     source          1..6991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        268..544
                     /label=chicken beta-actin promoter
     intron          545..1561
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1485..1507
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1569..1588
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1671..2378
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1816..1834)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2084..2103
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2482..2501)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2547..2602
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2601..2620)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   3273..3307
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3330..3346)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3330..3346)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3343..3365)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3354..3370
                     /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(3378..3384,3385..3402,3403..3408))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3423..3444
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3503..3698
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3549..3684
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3704..3838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3741..3760)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3795..3814
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3906..3923)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4077..4665)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4157..4176)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4836..5627,5628..5696))
                     /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     5459..5478
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5697..5801)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   5839..5901
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    6198..6231
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6392..6640
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6392..6412
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6563..6582
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    6737..6854
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        join(6954..6991,1..266)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
       61 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      121 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      181 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      241 acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc
      301 ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg
      361 cagcgatggg ggcggggggg ggggggggsg cgcgccaggg gggggggggg ggggggaggg
      421 gggggggggg gsgaggsgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      481 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      541 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      601 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      661 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      721 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      781 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      841 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
      901 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
      961 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1021 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1081 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1141 ggcggggccg ccycgggccg gggrgggctc gggggrgggg cgcggcggcc cccggagcgc
     1201 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1261 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1321 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1381 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1441 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1501 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca
     1561 gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattctgcag
     1621 tcgacggtac cgcgggcccg ctaccggact cagatctcga gctcaagctt atggtgagca
     1681 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     1741 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1801 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     1861 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     1921 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1981 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     2041 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     2101 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     2161 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     2221 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     2281 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     2341 gccgccactc caccggcggc atggacgagc tgtacaagtg ctgcatcatc ctgtaagcgg
     2401 ccgcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct
     2461 ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa
     2521 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     2581 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     2641 tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa
     2701 caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt
     2761 attccataga aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt
     2821 tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct
     2881 ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca
     2941 gagctccagc ctacacaatc gctcaagacg tgtaatgctt ttattatata ttagtcacga
     3001 tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac
     3061 aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt
     3121 ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc
     3181 ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct
     3241 atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt
     3301 tctagggtta aaagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3361 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3421 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3481 aacctgtcgt gccagcggat ccgcatctca ataagtcagc aaccatagtc ccgcccctaa
     3541 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3601 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     3661 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc
     3721 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3781 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg
     3841 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3901 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3961 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4021 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4081 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4141 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4201 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4261 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4321 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4381 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4441 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4501 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4561 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4621 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4681 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4741 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4801 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4861 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4921 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4981 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5041 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5101 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5161 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5221 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5281 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5341 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5401 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5461 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5521 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5581 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5641 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5701 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5761 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5821 ccacctgggt cgacttaacc ctagaaagat aatcatattg tgacgtacgt taaagataat
     5881 catgcgtaaa attgacgcat gtgttttatc gatctgtata tcgaggttta tttattaatt
     5941 tgaatagata ttaagtttta ttatatttac acttacatac taataataaa ttcaacaaac
     6001 aatttattta tgtttattta tttattaaaa aaaaacaaaa actcaaaatt tcttctataa
     6061 agtaacaaaa cttttaaaca ttctctcttt tacaaaaata aacttatttt gtactttaaa
     6121 aacagtcatg ttgtattata aaataagtaa ttagcttaac ttatacataa tagaaacaaa
     6181 ttatacttat taatcgcata acttcgtata atgtatgcta tacgaagtta tgcgattaag
     6241 ggatctgtag ggcgcagtag tccaactagt caggcccgaa ggaatagaag aagaaggtgg
     6301 agagagagac agagacagat ccattcgatt agtgaacgga tctcgacggt atcgatcacg
     6361 agactagcct cgagcggccg cccccttcac cgagggccta tttcccatga ttccttcata
     6421 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     6481 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     6541 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     6601 tttcttggct ttatatatct tgtggaaagg acgaaacacc gggcaagatt acaggagttt
     6661 caactcgagt tgaaactcct gtaatcttgc tttttgaatt ctcgacctcg agacaaatgg
     6721 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     6781 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     6841 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg cactagttat
     6901 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     6961 taacttacgg taaatggccc gcctggctga c
//
