LOCUS       sequence-456860-        6990 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Membrane tethered mCherry and scramble of shRNA targeting Atg7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6990)
  TITLE     Astrocytic LRRK2 Controls Synaptic Connectivity via Regulation of 
            ERM Phosphorylation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6990)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pPB-CAG-shControl-mCherry-CAAX
FEATURES             Location/Qualifiers
     source          1..6990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     286..308
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     370..389
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             472..1179
                     /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(617..635)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     885..904
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1283..1302)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1348..1403
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1402..1421)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   2074..2108
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2131..2147)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2131..2147)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2144..2166)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2155..2171
                     /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(2179..2185,2186..2203,2204..2209))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2224..2245
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2304..2499
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2350..2485
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2505..2639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2542..2561)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2596..2615
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2707..2724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2878..3466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2958..2977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3637..4428,4429..4497))
                     /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     4260..4279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4498..4602)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   4640..4702
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    4999..5032
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5193..5441
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5193..5213
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5364..5383
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    5538..5655
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        5755..6058
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6060..6335
                     /label=chicken beta-actin promoter
     intron          join(6336..6990,1..362)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
ORIGIN
        1 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
       61 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
      121 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
      181 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
      241 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
      301 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
      361 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattctgca
      421 gtcgacggta ccgcgggccc gctaccggac tcagatctcg agctcaagct tatggtgagc
      481 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
      541 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
      601 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
      661 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
      721 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
      781 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
      841 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
      901 aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
      961 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     1021 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag
     1081 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     1141 ggccgccact ccaccggcgg catggacgag ctgtacaagt gctgcatcat cctgtaagcg
     1201 gccgcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     1261 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     1321 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     1381 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     1441 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     1501 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     1561 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     1621 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     1681 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc
     1741 agagctccag cctacacaat cgctcaagac gtgtaatgct tttattatat attagtcacg
     1801 atatctataa caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa
     1861 caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat
     1921 tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg
     1981 cctcacggga gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc
     2041 tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct
     2101 ttctagggtt aaaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     2161 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     2221 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     2281 aaacctgtcg tgccagcgga tccgcatctc aataagtcag caaccatagt cccgccccta
     2341 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     2401 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     2461 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag
     2521 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2581 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc
     2641 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2701 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2761 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2821 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2881 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2941 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3001 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3061 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3121 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3181 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3241 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3301 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3361 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3421 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3481 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3541 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3601 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3661 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3721 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3781 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3841 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3901 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3961 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4021 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4081 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4141 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4201 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4261 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4321 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4381 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4441 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4501 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4561 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4621 gccacctggg tcgacttaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa
     4681 tcatgcgtaa aattgacgca tgtgttttat cgatctgtat atcgaggttt atttattaat
     4741 ttgaatagat attaagtttt attatattta cacttacata ctaataataa attcaacaaa
     4801 caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata
     4861 aagtaacaaa acttttaaac attctctctt ttacaaaaat aaacttattt tgtactttaa
     4921 aaacagtcat gttgtattat aaaataagta attagcttaa cttatacata atagaaacaa
     4981 attatactta ttaatcgcat aacttcgtat aatgtatgct atacgaagtt atgcgattaa
     5041 gggatctgta gggcgcagta gtccaactag tcaggcccga aggaatagaa gaagaaggtg
     5101 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac
     5161 gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat
     5221 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     5281 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     5341 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     5401 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggagtacta ttcataaccg
     5461 caacctcgag gttgcggtta tgaatagtac ttttttgaat tctcgacctc gagacaaatg
     5521 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     5581 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     5641 aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gcactagtta
     5701 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     5761 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     5821 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     5881 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     5941 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     6001 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     6061 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     6121 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
     6181 gcgcgccagg gggggggggg gggggggagg gggggggsgg ggsgaggsgg agaggtgcgg
     6241 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
     6301 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
     6361 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
     6421 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt
     6481 aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc
     6541 ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg
     6601 cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg
     6661 cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg
     6721 gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg
     6781 tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg
     6841 gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg
     6901 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggrgggct
     6961 cgggggaggg gcgcggcggc ccccggagcg
//
