LOCUS       sequence-456854-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To over express Phospho-mimetic Ezrin under gfaABC1D promoter in 
            astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6973)
  AUTHORS   Wang S, Baumert R, Sejourne G, Bindu DS, Dimond K, Sakers K, Vazquez
            L, Moore JL, Tan CX, Takano T, Rodriguez MP, Brose N, Bradley L, 
            Lessing R, Soderling SH, La Spada AR, Eroglu C
  TITLE     PD-linked LRRK2 G2019S mutation impairs astrocyte morphology and 
            synapse maintenance via ERM hyperphosphorylation
  JOURNAL   eLife 2025 [Reviewed Preprint] 14:RP107556. doi: 
            https://doi.org/10.7554/eLife.107556.1
REFERENCE   2  (bases 1 to 6973)
  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: "eLife 2025 
            [Reviewed Preprint] 14:RP107556. doi: 
            https://doi.org/10.7554/eLife.107556.1"
COMMENT     Sequence Label: pZac2.1-GfaABC1D-Ezrin T567D-BioID2-HA
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(453..1244,1245..1313))
                     /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"
     promoter        complement(1314..1418)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1444..1899)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1581..1602)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1793..1812
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2026..2048
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2040..2057
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2041..2057
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2076..2205
                     /label=AAV2 ITR
     polyA_signal    2367..2488
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2404..2423)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2458..2477
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(2528..2554)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2564..3262)
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="MFKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKW
                     LSQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKV
                     SGVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISE
                     NLKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKE
                     ILSGEFSLRRS"
     promoter        complement(5052..5070)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5113..5132)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          5115..5247
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     repeat_region   6098..6227
                     /label=AAV2 ITR
     primer_bind     complement(6265..6281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6265..6281)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6278..6300)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6289..6305
                     /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(6313..6319,6320..6337,6338..6343))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6358..6379
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(join(6667..6973,1..282))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6747..6766)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
       61 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      121 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      181 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
      241 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      301 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      361 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      421 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
      481 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
      541 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
      601 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
      661 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
      721 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
      781 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
      841 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
      901 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
      961 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1021 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1081 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1141 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1201 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1261 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1321 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1381 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1441 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     1501 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     1561 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     1621 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     1681 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     1741 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     1801 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     1861 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     1921 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     1981 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     2041 gtaaaacgac ggccagtgaa ttaggttaat taaggctgcg cgctcgctcg ctcactgagg
     2101 ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc
     2161 gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag ttaatgatta
     2221 acccgccatg ctacttatct acgtagccat gctctaggaa gatccttatc gattttacca
     2281 catttgtaga ggttttactt gctttaaaaa acctcccaca tctccccctg aacctgaaac
     2341 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     2401 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2461 gtttgtccaa actcatcaat gtatcttatc atgtctgctc gaagcggccg cccgggtcga
     2521 ctctagatgc gtaatccggt acatcgtaag ggtaagcgct agctgaccgt cgcaagctaa
     2581 attcgccgga cagtatctcc tttatgcctt cctctgtaag tatcaaagca ccgccctttt
     2641 cactcaggcc cacaagtttc ccggtaatct tgccttctcc caagagttta acctcctctc
     2701 caaggtacaa catctttgac tcaatttttc ctttgaactc tttaaaggat ttttccttaa
     2761 atttcttgag attttcggaa attcgcttga gaaccttcag caagacctcc tttctgtccc
     2821 aatcctttcc agtgatttca taaagtgttg tggcccgatc cttgatctct tcagggatct
     2881 ctctttgatt cacgtttata ccgattccca ctatcagctt gtccttactg agctcacaaa
     2941 ggacccctga aacctttttc tcctgaaaat aaacgtcgtt gggccacttc agactaaatg
     3001 gtatctctgt aatctcttcg agtgcctcgc tcacggagag tcccaagacc agtggaagtt
     3061 gcaacaaatt ttcaaactct tttggattca acaagaaaga gaagtaaaga ccgccctcct
     3121 gactcaacca cttccgtccg agaccgcctc tacctttggt ctgtctatcg gcgacgagtg
     3181 cagtgccata ggagacattc cattccttaa gccgctcctg ggtgctatca acttccttca
     3241 gccaaataag atttttgaac atggtgaatt ccgacagggc ctcgaactcg tcgatgcgct
     3301 gcttggtgtt gccctgccgg atctgccgca ggtccttgta cttgtcccgg ccttgcctca
     3361 tgttctcgtt gtggatgatg tcattgtggg tcctcttatt ctcatctcgg gcctgggaca
     3421 gctcgctgct cagcgtcagc agctgccgct gcacacgctc gttcttctct gcctcagtga
     3481 tgcgcttctc ctcattgcgg tcatcccgga tgccctcact agacagctcc gcgctgtagc
     3541 ccgtgggctc tgcgccctca tcctgcaagc tctcctggac atggtagctc accggctcgt
     3601 acacgggggg tggtgggggc gggggtgctg tcatcaccag gtgcagctcc tccttggtct
     3661 tcaccaggtc atcctgggct tctttggccc tgtgctgcca ctcttcaact tcatcctcct
     3721 tgcgcctccg cgcctcttcc aggagggcaa tcttggcagt gtattctgca agctccgcag
     3781 ccagctgctc ctggctcttt atctgatcca ccgcctgtct ctccagctcc tccttagccc
     3841 gcagtgcagc catacggtca gcctctaggc gctcggcctc ctcctgtgcc cgcttcctct
     3901 cctcctccag ctgcagggcc ctctgaatct gctccgagag ctctctctct gccttctttg
     3961 tcttctcctc atagtcctgc agccgcagca tcaactcctc cttctcgcgc atcatctgct
     4021 ctttctctct ctccacggtt tctctccttt tcttctctgt ttccagctgt tgccgctcca
     4081 gctgcttctg atgcttctcc tcccgggcct gggccttcat ctgctgcacc tcgatggtgt
     4141 caggcttcct gcggcgcata tacaactcat ggttgcccat gcagagctgc aggatccgct
     4201 tgttgattct cagacgtggg gcataaaaca caaagtcagg tgccttcttg tcgatgggtt
     4261 taatgacaaa ctttttgtca ttgaaagaga tgttcctgat ttcactccaa ggaaagccaa
     4321 tctttggggt taacttatca tctttctcat aaatattcag tccaagggca tcaactccaa
     4381 gccaaaggtc tgttcctttc ttgtttttta tctcgaaata gttgattcca tacatttcca
     4441 ggtcctgagc aatcttcagg tattccaaca tagcattatc tttgagcatc ccacggtgtt
     4501 ccgcatgcca cacctggatc cggtcctccc actggtccct ggtaagtttg tgctggtcca
     4561 tcactctttg agggatcagc cgctcagagc tgaggtaccc agacttgtgc acttctttgt
     4621 tgtagtcccc aaacttggcc tgcacagcgt aggaccccaa gagcacggca gtctcagggg
     4681 ggcagtagat ctcatcgcta aggattcctt ccttcacttg gaggaagaaa agtttctggg
     4741 tgatgtcctg gatgagctcc tcagccacat cttcagggta gaacttggcc cggaacttga
     4801 actggagggg attctccttc ctgacctcct gggcagacac cttcttatcc agcttcagcc
     4861 aggtaggaaa tcctttatta tccacatagt ggaggccaaa gtaccacact tcccggaggc
     4921 cgatagtctt taccacctga tcaaaaagct gttttccagt tgtatttggc tggattgcaa
     4981 actccagctc tgcatccatg gtggtaactc ggacattgat tggtttcggc atggtgaatt
     5041 ctcgaggcta gcctatagtg agtcgtatta agtactctag ccttaagagc tgtaattgaa
     5101 ctgggagtgg acacctgtgg agagaaaggc aaagtggatg tcagtaagac caataggtgc
     5161 ctatcagaaa cgcaagagtc ttctctgtct cgacaagccc agtttctatt ggtctcctta
     5221 aacctgtctt gtaaccttga tacttacctg cccagtgcct cacgaccaac ttctgcagct
     5281 taagttcgag actgttgtgt cagaagcact gactgcgtta gcaatttaac tgtgataaac
     5341 taccgcaata aagcttgcga gcagcggagg tgatgcgtct cctctccaac ctgctctggc
     5401 tctgctcgct cctgggatgc gagggcttta tgaaggagtg ggctagactg gcgatgcccg
     5461 ggtgcccctg gcaacacccc ctggcatagc ctgagggggt ggggttgggc cgcagcccag
     5521 ctatggggag agctctcccc tcacccattt gtgtccttgg acttctggga agcagacagc
     5581 cctcctctcc ccccctgcca cccactgtcc atgcctgggc atcccctgat cccctttcct
     5641 gggcacaggc tgaatagagc cttgttctcc accgccggtg gcgccacaca gcaagggcag
     5701 cccctagggg gctgtgctgc ttttatccca agatgccagg ctgtcaggct ggggtgcaat
     5761 gcaagccccc tgctcaatgg gcttctcgga aggcatttga ggggggctgg ggtgcggcgg
     5821 gaagcaggca ctgtgcccac cccgaacatt gtgtctgtgc caaggtgagt cattcactgg
     5881 gcatgaagag gaggccctgg ggggcggcca gcccaatgct gccaggtctg cttgcagaca
     5941 aggcctggct gtctgcctcc tccccagctc acagagccag ctcaggcccc cgagcctctg
     6001 tcagagcagc gtcccctact ccacaccagg atatgttaga tcttcctaga gcatggctac
     6061 gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg
     6121 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     6181 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagcct aattaaggcc
     6241 ttaattaaat ctggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     6301 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6361 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6421 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6481 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6541 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6601 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6661 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6721 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6781 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6841 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6901 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6961 cggtaactat cgt
//
