LOCUS       sequence-456695-        6990 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Membrane tethered mCherry and shRNA targeting LRRK2.
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  JOURNAL   Unpublished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            "Unpublished"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-shLRRK2-mCherry-CAAX
FEATURES             Location/Qualifiers
     source          1..6990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(355..374)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    420..475
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(474..493)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   1146..1180
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1203..1219)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1203..1219)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1216..1238)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1227..1243
                     /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(1251..1257,1258..1275,1276..1281))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1296..1317
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1376..1571
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1422..1557
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1577..1711
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1614..1633)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1668..1687
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1779..1796)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1950..2538)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2030..2049)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2709..3500,3501..3569))
                     /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     3332..3351
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3570..3674)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   3712..3774
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    4071..4104
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4265..4513
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4265..4285
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4436..4455
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    4610..4727
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        4827..5130
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5132..5407
                     /label=chicken beta-actin promoter
     intron          5408..6424
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     6348..6370
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     6432..6451
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(6534..6990,1..251)
                     /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(6679..6697)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6947..6966
                     /label=mCherry-F
                     /note="mCherry, forward primer"
ORIGIN
        1 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
       61 aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc
      121 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
      181 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
      241 agctgtacaa gtgctgcatc atcctgtaag cggccgcact cctcaggtgc aggctgccta
      301 tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact gagatctttt
      361 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta
      421 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg
      481 aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt
      541 ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca
      601 gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg
      661 ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc
      721 cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt
      781 ccctcttctc ttatggagat ccctcgacct gcagagctcc agcctacaca atcgctcaag
      841 acgtgtaatg cttttattat atattagtca cgatatctat aacaagaaaa tatatatata
      901 ataagttatc acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct
      961 taaaagtcac gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa
     1021 aatcagtgac acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga
     1081 gatgtcctaa atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa
     1141 gaatgcatgc gtcaatttta cgcagactat ctttctaggg ttaaaagctt ggcgtaatca
     1201 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     1261 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1321 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc
     1381 tcaataagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     1441 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     1501 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     1561 gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1621 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1681 actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg
     1741 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     1801 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1861 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1921 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1981 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2041 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2101 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2161 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2221 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2281 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2341 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2401 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2461 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2521 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2581 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2641 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2701 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2761 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2821 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     2881 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2941 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3001 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3061 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3121 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3181 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3241 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3301 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3361 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3421 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3481 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3541 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3601 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3661 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ggtcgactta accctagaaa
     3721 gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg catgtgtttt
     3781 atcgatctgt atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt
     3841 tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta
     3901 aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttaa acattctctc
     3961 ttttacaaaa ataaacttat tttgtacttt aaaaacagtc atgttgtatt ataaaataag
     4021 taattagctt aacttataca taatagaaac aaattatact tattaatcgc ataacttcgt
     4081 ataatgtatg ctatacgaag ttatgcgatt aagggatctg tagggcgcag tagtccaact
     4141 agtcaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
     4201 attagtgaac ggatctcgac ggtatcgatc acgagactag cctcgagcgg ccgccccctt
     4261 caccgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     4321 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     4381 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     4441 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     4501 aggacgaaac accggggccg agttgtggat catattctcg agaatatgat ccacaactcg
     4561 gcctttttga attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa
     4621 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
     4681 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
     4741 gggacagcag agatccactt tggcactagt tattaatagt aatcaattac ggggtcatta
     4801 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     4861 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     4921 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     4981 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     5041 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     5101 atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact
     5161 ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
     5221 tgtgcagcga tgggggcggg gggggggggg gggcgcgcca gggggggggg ggggggggga
     5281 gggggggggs ggggsgaggs ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg
     5341 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg
     5401 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg
     5461 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc
     5521 ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct
     5581 gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg
     5641 tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg
     5701 tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc
     5761 ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg
     5821 gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc
     5881 cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac
     5941 ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg
     6001 cggggcgggg ccgcctcggg ccggggrggg ctcgggggag gggcgcggcg gcccccggag
     6061 cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga
     6121 gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg
     6181 ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg
     6241 cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg
     6301 ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg
     6361 gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct
     6421 acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattctg
     6481 cagtcgacgg taccgcgggc ccgctaccgg actcagatct cgagctcaag cttatggtga
     6541 gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca
     6601 tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     6661 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct
     6721 gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg
     6781 acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     6841 acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt
     6901 tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga
     6961 agaagaccat gggctgggag gcctcctccg
//
