LOCUS       sequence-459562-        8743 bp    DNA     circular SYN 15-APR-2025
DEFINITION  To over express 2x Ezrin under gfaABC1D promoter in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8743)
  TITLE     Astrocytic LRRK2 Controls Synaptic Connectivity via Regulation of 
            ERM Phosphorylation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8743)
  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            "Unpublished"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-2xEzrin-BioID2-HA
FEATURES             Location/Qualifiers
     source          1..8743
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     intron          1239..1371
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1354..1373
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1416..1434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4994..5692
                     /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             5702..5728
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5768..5889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5779..5798)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5833..5852
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6051..6180
                     /label=AAV2 ITR
     primer_bind     complement(6199..6216)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6199..6215)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6208..6230)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6357..6812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6444..6463)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6654..6675
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6838..6942
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6943..7011,7012..7803)
                     /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      7974..8562
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8463..8482
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatctaa catatcctgg tgtggagtag gggacgctgc
      481 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      541 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      601 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      661 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      721 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      781 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      841 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      901 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      961 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
     1021 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga
     1081 gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca agctttattg
     1141 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
     1201 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     1261 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     1321 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     1381 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     1441 tcgagaattc accatgccga aaccaatcaa tgtccgagtt accaccatgg atgcagagct
     1501 ggagtttgca atccagccaa atacaactgg aaaacagctt tttgatcagg tggtaaagac
     1561 tatcggcctc cgggaagtgt ggtactttgg cctccactat gtggataata aaggatttcc
     1621 tacctggctg aagctggata agaaggtgtc tgcccaggag gtcaggaagg agaatcccct
     1681 ccagttcaag ttccgggcca agttctaccc tgaagatgtg gctgaggagc tcatccagga
     1741 catcacccag aaacttttct tcctccaagt gaaggaagga atccttagcg atgagatcta
     1801 ctgcccccct gagactgccg tgctcttggg gtcctacgct gtgcaggcca agtttgggga
     1861 ctacaacaaa gaagtgcaca agtctgggta cctcagctct gagcggctga tccctcaaag
     1921 agtgatggac cagcacaaac ttaccaggga ccagtgggag gaccggatcc aggtgtggca
     1981 tgcggaacac cgtgggatgc tcaaagataa tgctatgttg gaatacctga agattgctca
     2041 ggacctggaa atgtatggaa tcaactattt cgagataaaa aacaagaaag gaacagacct
     2101 ttggcttgga gttgatgccc ttggactgaa tatttatgag aaagatgata agttaacccc
     2161 aaagattggc tttccttgga gtgaaatcag gaacatctct ttcaatgaca aaaagtttgt
     2221 cattaaaccc atcgacaaga aggcacctga ctttgtgttt tatgccccac gtctgagaat
     2281 caacaagcgg atcctgcagc tctgcatggg caaccatgag ttgtatatgc gccgcaggaa
     2341 gcctgacacc atcgaggtgc agcagatgaa ggcccaggcc cgggaggaga agcatcagaa
     2401 gcagctggag cggcaacagc tggaaacaga gaagaaaagg agagaaaccg tggagagaga
     2461 gaaagagcag atgatgcgcg agaaggagga gttgatgctg cggctgcagg actatgagga
     2521 gaagacaaag aaggcagaga gagagctctc ggagcagatt cagagggccc tgcagctgga
     2581 ggaggagagg aagcgggcac aggaggaggc cgagcgccta gaggctgacc gtatggctgc
     2641 actgcgggct aaggaggagc tggagagaca ggcggtggat cagataaaga gccaggagca
     2701 gctggctgcg gagcttgcag aatacactgc caagattgcc ctcctggaag aggcgcggag
     2761 gcgcaaggag gatgaagttg aagagtggca gcacagggcc aaagaagccc aggatgacct
     2821 ggtgaagacc aaggaggagc tgcacctggt gatgacagca cccccgcccc caccaccccc
     2881 cgtgtacgag ccggtgagct accatgtcca ggagagcttg caggatgagg gcgcagagcc
     2941 cacgggctac agcgcggagc tgtctagtga gggcatccgg gatgaccgca atgaggagaa
     3001 gcgcatcact gaggcagaga agaacgagcg tgtgcagcgg cagctgctga cgctgagcag
     3061 cgagctgtcc caggcccgag atgagaataa gaggacccac aatgacatca tccacaacga
     3121 gaacatgagg caaggccggg acaagtacaa gacgctgcgg cagatccggc agggcaacac
     3181 caagcagcgc atcgacgagt tcgaggccct gtcggaattc accatgccga aaccaatcaa
     3241 tgtccgagtt accaccatgg atgcagagct ggagtttgca atccagccaa atacaactgg
     3301 aaaacagctt tttgatcagg tggtaaagac tatcggcctc cgggaagtgt ggtactttgg
     3361 cctccactat gtggataata aaggatttcc tacctggctg aagctggata agaaggtgtc
     3421 tgcccaggag gtcaggaagg agaatcccct ccagttcaag ttccgggcca agttctaccc
     3481 tgaagatgtg gctgaggagc tcatccagga catcacccag aaacttttct tcctccaagt
     3541 gaaggaagga atccttagcg atgagatcta ctgcccccct gagactgccg tgctcttggg
     3601 gtcctacgct gtgcaggcca agtttgggga ctacaacaaa gaagtgcaca agtctgggta
     3661 cctcagctct gagcggctga tccctcaaag agtgatggac cagcacaaac ttaccaggga
     3721 ccagtgggag gaccggatcc aggtgtggca tgcggaacac cgtgggatgc tcaaagataa
     3781 tgctatgttg gaatacctga agattgctca ggacctggaa atgtatggaa tcaactattt
     3841 cgagataaaa aacaagaaag gaacagacct ttggcttgga gttgatgccc ttggactgaa
     3901 tatttatgag aaagatgata agttaacccc aaagattggc tttccttgga gtgaaatcag
     3961 gaacatctct ttcaatgaca aaaagtttgt cattaaaccc atcgacaaga aggcacctga
     4021 ctttgtgttt tatgccccac gtctgagaat caacaagcgg atcctgcagc tctgcatggg
     4081 caaccatgag ttgtatatgc gccgcaggaa gcctgacacc atcgaggtgc agcagatgaa
     4141 ggcccaggcc cgggaggaga agcatcagaa gcagctggag cggcaacagc tggaaacaga
     4201 gaagaaaagg agagaaaccg tggagagaga gaaagagcag atgatgcgcg agaaggagga
     4261 gttgatgctg cggctgcagg actatgagga gaagacaaag aaggcagaga gagagctctc
     4321 ggagcagatt cagagggccc tgcagctgga ggaggagagg aagcgggcac aggaggaggc
     4381 cgagcgccta gaggctgacc gtatggctgc actgcgggct aaggaggagc tggagagaca
     4441 ggcggtggat cagataaaga gccaggagca gctggctgcg gagcttgcag aatacactgc
     4501 caagattgcc ctcctggaag aggcgcggag gcgcaaggag gatgaagttg aagagtggca
     4561 gcacagggcc aaagaagccc aggatgacct ggtgaagacc aaggaggagc tgcacctggt
     4621 gatgacagca cccccgcccc caccaccccc cgtgtacgag ccggtgagct accatgtcca
     4681 ggagagcttg caggatgagg gcgcagagcc cacgggctac agcgcggagc tgtctagtga
     4741 gggcatccgg gatgaccgca atgaggagaa gcgcatcact gaggcagaga agaacgagcg
     4801 tgtgcagcgg cagctgctga cgctgagcag cgagctgtcc caggcccgag atgagaataa
     4861 gaggacccac aatgacatca tccacaacga gaacatgagg caaggccggg acaagtacaa
     4921 gacgctgcgg cagatccggc agggcaacac caagcagcgc atcgacgagt tcgaggccct
     4981 gtcggaattc accatgttca aaaatcttat ttggctgaag gaagttgata gcacccagga
     5041 gcggcttaag gaatggaatg tctcctatgg cactgcactc gtcgccgata gacagaccaa
     5101 aggtagaggc ggtctcggac ggaagtggtt gagtcaggag ggcggtcttt acttctcttt
     5161 cttgttgaat ccaaaagagt ttgaaaattt gttgcaactt ccactggtct tgggactctc
     5221 cgtgagcgag gcactcgaag agattacaga gataccattt agtctgaagt ggcccaacga
     5281 cgtttatttt caggagaaaa aggtttcagg ggtcctttgt gagctcagta aggacaagct
     5341 gatagtggga atcggtataa acgtgaatca aagagagatc cctgaagaga tcaaggatcg
     5401 ggccacaaca ctttatgaaa tcactggaaa ggattgggac agaaaggagg tcttgctgaa
     5461 ggttctcaag cgaatttccg aaaatctcaa gaaatttaag gaaaaatcct ttaaagagtt
     5521 caaaggaaaa attgagtcaa agatgttgta ccttggagag gaggttaaac tcttgggaga
     5581 aggcaagatt accgggaaac ttgtgggcct gagtgaaaag ggcggtgctt tgatacttac
     5641 agaggaaggc ataaaggaga tactgtccgg cgaatttagc ttgcgacggt cagctagcgc
     5701 ttacccttac gatgtaccgg attacgcatc tagagtcgac ccgggcggcc gcttcgagca
     5761 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     5821 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     5881 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg
     5941 gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatcttcct
     6001 agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct
     6061 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     6121 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     6181 ccttaattaa cctaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     6241 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     6301 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     6361 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     6421 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     6481 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     6541 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     6601 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     6661 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     6721 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     6781 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     6841 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     6901 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6961 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     7021 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     7081 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     7141 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     7201 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     7261 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     7321 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     7381 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     7441 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     7501 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     7561 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     7621 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     7681 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     7741 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     7801 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     7861 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     7921 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7981 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     8041 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     8101 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     8161 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     8221 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     8281 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     8341 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     8401 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     8461 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     8521 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     8581 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     8641 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     8701 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aag
//
