LOCUS       sequence-363021-        6299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Ezr sgRNAs under the U6 promoter and mcherry protein 
            specifically in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6299)
  AUTHORS   Endo F, Kasai A, Soto JS, Yu X, Qu Z, Hashimoto H, Gradinaru V, 
            Kawaguchi R, Khakh BS
  TITLE     Molecular basis of astrocyte diversity and morphology across the CNS
            in health and disease.
  JOURNAL   Science. 2022 Nov 4;378(6619):eadc9020. doi: 
            10.1126/science.adc9020. Epub 2022 Nov 4.
  PUBMED    36378959
REFERENCE   2  (bases 1 to 6299)
  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: "Science."; 
            date: "2022-11-4"; volume: "378(6619):eadc9020. doi"; pages: " 
            10.1126/science.adc9020. Epub 2022 Nov 4"
COMMENT     Sequence Label: Pzac2.1 U6-Ezr sgRNA1, 2, 3_gfaABC1D mcherry SV40
FEATURES             Location/Qualifiers
     source          1..6299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(143..161)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    747..868
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(758..777)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     812..831
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   1030..1159
                     /label=AAV2 ITR
     primer_bind     complement(1178..1195)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1178..1194)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1187..1209)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1336..1791
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1423..1442)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1633..1654
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1817..1921
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1922..1990,1991..2782)
                     /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      2953..3541
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3442..3461
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3829..3850
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3865..3870,3871..3888,3889..3895)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3903..3919
                     /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     3908..3930
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3927..3943
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3927..3943
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   3981..4110
                     /label=AAV2 ITR
     promoter        4174..4422
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        4444..4519
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        4541..4789
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        4812..4887
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        4909..5157
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        5180..5255
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     intron          6079..6211
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     6194..6213
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        6256..6274
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(6297..6299,1..708)
                     /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"
ORIGIN
        1 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
       61 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
      121 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
      181 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
      241 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
      301 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
      361 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
      421 cagaagaaaa ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
      481 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
      541 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
      601 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
      661 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaact agagtcgacc
      721 cgggcggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
      781 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
      841 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
      901 ggttcagggg gagatgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa
      961 aatcgataag gatcttccta gagcatggct acgtagataa gtagcatggc gggttaatca
     1021 ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     1081 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     1141 tgagcgagcg agcgcgcagc cttaattaac ctaattcact ggccgtcgtt ttacaacgtc
     1201 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     1261 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     1321 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     1381 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     1441 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1501 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     1561 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1621 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1681 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1741 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     1801 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1861 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     1921 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     1981 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2041 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2101 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2161 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2221 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2281 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2341 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2401 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2461 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2521 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2581 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     2641 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2701 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2761 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     2821 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     2881 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2941 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3001 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3061 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3121 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3181 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3241 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3301 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3361 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3421 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3481 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3541 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3601 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3661 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3721 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3781 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     3841 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     3901 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccagatt
     3961 taattaaggc cttaattagg ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc
     4021 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg
     4081 gagtggccaa ctccatcact aggggttcct tgtagttaat gattaacccg ccatgctact
     4141 tatctacgta gccatgctct aggaagatct accgagggcc tatttcccat gattccttca
     4201 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
     4261 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     4321 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     4381 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggtgtggc acgcggaaca
     4441 ccggttttag tactctggaa acagaatcta ctaaaacaag gcaaaatgcc gtgtttatct
     4501 cgtcaacttg ttggcgagat ttttgcggcc gcgatctacc gagggcctat ttcccatgat
     4561 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
     4621 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     4681 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     4741 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg agcttctgtg
     4801 tgatgtcctg ggttttagta ctctggaaac agaatctact aaaacaaggc aaaatgccgt
     4861 gtttatctcg tcaacttgtt ggcgagattt ttgcggccgc gatctaccga gggcctattt
     4921 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
     4981 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     5041 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     5101 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgtg
     5161 accaccatgg atgccgagcg ttttagtact ctggaaacag aatctactaa aacaaggcaa
     5221 aatgccgtgt ttatctcgtc aacttgttgg cgagattttt gcggccgcct agaggtaccc
     5281 gttgatctaa catatcctgg tgtggagtag gggacgctgc tctgacagag gctcgggggc
     5341 ctgagctggc tctgtgagct ggggaggagg cagacagcca ggccttgtct gcaagcagac
     5401 ctggcagcat tgggctggcc gccccccagg gcctcctctt catgcccagt gaatgactca
     5461 ccttggcaca gacacaatgt tcggggtggg cacagtgcct gcttcccgcc gcaccccagc
     5521 ccccctcaaa tgccttccga gaagcccatt gagcaggggg cttgcattgc accccagcct
     5581 gacagcctgg catcttggga taaaagcagc acagccccct aggggctgcc cttgctgtgt
     5641 ggcgccaccg gcggtggaga acaaggctct attcagcctg tgcccaggaa aggggatcag
     5701 gggatgccca ggcatggaca gtgggtggca gggggggaga ggagggctgt ctgcttccca
     5761 gaagtccaag gacacaaatg ggtgagggga gagctctccc catagctggg ctgcggccca
     5821 accccacccc ctcaggctat gccagggggt gttgccaggg gcacccgggc atcgccagtc
     5881 tagcccactc cttcataaag ccctcgcatc ccaggagcga gcagagccag agcaggttgg
     5941 agaggagacg catcacctcc gctgctcgca agctttattg cggtagttta tcacagttaa
     6001 attgctaacg cagtcagtgc ttctgacaca acagtctcga acttaagctg cagaagttgg
     6061 tcgtgaggca ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag
     6121 aaactgggct tgtcgagaca gagaaaactc ttgcgtttct gataggcacc tattggtctt
     6181 actgacatcc actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct
     6241 taaggctaga gtacttaata cgactcacta taggctagcc tcgagaattc accaccatg
//
