LOCUS       sequence-456856-        6973 bp    DNA     circular SYN 15-APR-2025
DEFINITION  To over express Phospho-dead Ezrin under gfaABC1D promoter in 
            astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6973)
  TITLE     Astrocytic LRRK2 Controls Synaptic Connectivity via Regulation of 
            ERM Phosphorylation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6973)
  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            "Unpublished"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: pZac2.1-GfaABC1D-Ezrin T567A-BioID2-HA
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          671..803
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     786..805
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        848..866
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2656..3354
                     /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"
     CDS             3364..3390
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3430..3551
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3441..3460)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3495..3514
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3713..3842
                     /label=AAV2 ITR
     primer_bind     complement(3861..3878)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3861..3877)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3870..3892)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4019..4474
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4106..4125)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4316..4337
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4500..4604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4605..4673,4674..5465)
                     /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"
     rep_origin      5636..6224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6125..6144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6512..6533
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6548..6553,6554..6571,6572..6578)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6586..6602
                     /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)."
     primer_bind     6591..6613
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6610..6626
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6610..6626
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   6664..6793
                     /label=AAV2 ITR
ORIGIN
        1 attgggctgg ccgcccccca gggcctcctc ttcatgccca gtgaatgact caccttggca
       61 cagacacaat gttcggggtg ggcacagtgc ctgcttcccg ccgcacccca gcccccctca
      121 aatgccttcc gagaagccca ttgagcaggg ggcttgcatt gcaccccagc ctgacagcct
      181 ggcatcttgg gataaaagca gcacagcccc ctaggggctg cccttgctgt gtggcgccac
      241 cggcggtgga gaacaaggct ctattcagcc tgtgcccagg aaaggggatc aggggatgcc
      301 caggcatgga cagtgggtgg caggggggga gaggagggct gtctgcttcc cagaagtcca
      361 aggacacaaa tgggtgaggg gagagctctc cccatagctg ggctgcggcc caaccccacc
      421 ccctcaggct atgccagggg gtgttgccag gggcacccgg gcatcgccag tctagcccac
      481 tccttcataa agccctcgca tcccaggagc gagcagagcc agagcaggtt ggagaggaga
      541 cgcatcacct ccgctgctcg caagctttat tgcggtagtt tatcacagtt aaattgctaa
      601 cgcagtcagt gcttctgaca caacagtctc gaacttaagc tgcagaagtt ggtcgtgagg
      661 cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg
      721 cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat
      781 ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct cttaaggcta
      841 gagtacttaa tacgactcac tataggctag cctcgagaat tcaccatgcc gaaaccaatc
      901 aatgtccgag ttaccaccat ggatgcagag ctggagtttg caatccagcc aaatacaact
      961 ggaaaacagc tttttgatca ggtggtaaag actatcggcc tccgggaagt gtggtacttt
     1021 ggcctccact atgtggataa taaaggattt cctacctggc tgaagctgga taagaaggtg
     1081 tctgcccagg aggtcaggaa ggagaatccc ctccagttca agttccgggc caagttctac
     1141 cctgaagatg tggctgagga gctcatccag gacatcaccc agaaactttt cttcctccaa
     1201 gtgaaggaag gaatccttag cgatgagatc tactgccccc ctgagactgc cgtgctcttg
     1261 gggtcctacg ctgtgcaggc caagtttggg gactacaaca aagaagtgca caagtctggg
     1321 tacctcagct ctgagcggct gatccctcaa agagtgatgg accagcacaa acttaccagg
     1381 gaccagtggg aggaccggat ccaggtgtgg catgcggaac accgtgggat gctcaaagat
     1441 aatgctatgt tggaatacct gaagattgct caggacctgg aaatgtatgg aatcaactat
     1501 ttcgagataa aaaacaagaa aggaacagac ctttggcttg gagttgatgc ccttggactg
     1561 aatatttatg agaaagatga taagttaacc ccaaagattg gctttccttg gagtgaaatc
     1621 aggaacatct ctttcaatga caaaaagttt gtcattaaac ccatcgacaa gaaggcacct
     1681 gactttgtgt tttatgcccc acgtctgaga atcaacaagc ggatcctgca gctctgcatg
     1741 ggcaaccatg agttgtatat gcgccgcagg aagcctgaca ccatcgaggt gcagcagatg
     1801 aaggcccagg cccgggagga gaagcatcag aagcagctgg agcggcaaca gctggaaaca
     1861 gagaagaaaa ggagagaaac cgtggagaga gagaaagagc agatgatgcg cgagaaggag
     1921 gagttgatgc tgcggctgca ggactatgag gagaagacaa agaaggcaga gagagagctc
     1981 tcggagcaga ttcagagggc cctgcagctg gaggaggaga ggaagcgggc acaggaggag
     2041 gccgagcgcc tagaggctga ccgtatggct gcactgcggg ctaaggagga gctggagaga
     2101 caggcggtgg atcagataaa gagccaggag cagctggctg cggagcttgc agaatacact
     2161 gccaagattg ccctcctgga agaggcgcgg aggcgcaagg aggatgaagt tgaagagtgg
     2221 cagcacaggg ccaaagaagc ccaggatgac ctggtgaaga ccaaggagga gctgcacctg
     2281 gtgatgacag cacccccgcc cccaccaccc cccgtgtacg agccggtgag ctaccatgtc
     2341 caggagagct tgcaggatga gggcgcagag cccacgggct acagcgcgga gctgtctagt
     2401 gagggcatcc gggatgaccg caatgaggag aagcgcatca ctgaggcaga gaagaacgag
     2461 cgtgtgcagc ggcagctgct gacgctgagc agcgagctgt cccaggcccg agatgagaat
     2521 aagaggaccc acaatgacat catccacaac gagaacatga ggcaaggccg ggacaagtac
     2581 aaggcgctgc ggcagatccg gcagggcaac accaagcagc gcatcgacga gttcgaggcc
     2641 ctgtcggaat tcaccatgtt caaaaatctt atttggctga aggaagttga tagcacccag
     2701 gagcggctta aggaatggaa tgtctcctat ggcactgcac tcgtcgccga tagacagacc
     2761 aaaggtagag gcggtctcgg acggaagtgg ttgagtcagg agggcggtct ttacttctct
     2821 ttcttgttga atccaaaaga gtttgaaaat ttgttgcaac ttccactggt cttgggactc
     2881 tccgtgagcg aggcactcga agagattaca gagataccat ttagtctgaa gtggcccaac
     2941 gacgtttatt ttcaggagaa aaaggtttca ggggtccttt gtgagctcag taaggacaag
     3001 ctgatagtgg gaatcggtat aaacgtgaat caaagagaga tccctgaaga gatcaaggat
     3061 cgggccacaa cactttatga aatcactgga aaggattggg acagaaagga ggtcttgctg
     3121 aaggttctca agcgaatttc cgaaaatctc aagaaattta aggaaaaatc ctttaaagag
     3181 ttcaaaggaa aaattgagtc aaagatgttg taccttggag aggaggttaa actcttggga
     3241 gaaggcaaga ttaccgggaa acttgtgggc ctgagtgaaa agggcggtgc tttgatactt
     3301 acagaggaag gcataaagga gatactgtcc ggcgaattta gcttgcgacg gtcagctagc
     3361 gcttaccctt acgatgtacc ggattacgca tctagagtcg acccgggcgg ccgcttcgag
     3421 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     3481 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     3541 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt
     3601 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatcttc
     3661 ctagagcatg gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc
     3721 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     3781 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     3841 agccttaatt aacctaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     3901 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     3961 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     4021 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4081 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4141 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4201 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4261 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4321 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4381 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4441 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     4501 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4561 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4621 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4681 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4741 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4801 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4861 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4921 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4981 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5041 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5101 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5161 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5221 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5281 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5341 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5401 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5461 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5521 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5581 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5641 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5701 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5761 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5821 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5881 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5941 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6001 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6061 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6121 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6181 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6241 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6301 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6361 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     6421 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     6481 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     6541 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     6601 aatttcacac aggaaacagc tatgaccatg attacgccag atttaattaa ggccttaatt
     6661 aggctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
     6721 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
     6781 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
     6841 tctaggaaga tctaacatat cctggtgtgg agtaggggac gctgctctga cagaggctcg
     6901 ggggcctgag ctggctctgt gagctgggga ggaggcagac agccaggcct tgtctgcaag
     6961 cagacctggc agc
//
