LOCUS       sequence-390982-        5622 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the knock down of GFP with an astrocyte specific mCherry 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5622)
  AUTHORS   Ollivier M, Soto JS, Linker KE, Moye SL, Jami-Alahmadi Y, Jones AE, 
            Divakaruni AS, Kawaguchi R, Wohlschlegel JA, Khakh BS
  TITLE     Crym-positive striatal astrocytes gate perseverative behaviour.
  JOURNAL   Nature. 2024 Mar;627(8003):358-366. doi: 10.1038/s41586-024-07138-0.
            Epub 2024 Feb 28.
  PUBMED    38418885
REFERENCE   2  (bases 1 to 5622)
  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"; doi: 
            "10.1038/s41586-024-07138-0"; journalName: "Nature"; date: 
            "2024-03"; volume: "627"; issue: "8003"; pages: "358-366"
COMMENT     Sequence Label: U6-sgRNA-GFP
FEATURES             Location/Qualifiers
     source          1..5622
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     217..236
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    604..625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(640..645,646..663,664..670)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    678..694
                     /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     683..705
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     702..718
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     702..718
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   756..885
                     /label=AAV2 ITR
     primer_bind     1107..1126
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1206..1281
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     intron          2177..2309
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2292..2311
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        2354..2372
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2395..3105
                     /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(2540..2558)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3144..3265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3155..3174)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3209..3228
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3427..3556
                     /label=AAV2 ITR
     primer_bind     complement(3575..3592)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3575..3591)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3584..3606)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3733..4188
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3820..3839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4030..4051
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4214..4318
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4319..4387,4388..5179)
                     /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      join(5350..5622,1..316)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
       61 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
      121 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
      181 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
      241 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
      301 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
      361 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
      421 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
      481 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
      541 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      601 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      661 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      721 tgattacgcc agatttaatt aaggccttaa ttaggctgcg cgctcgctcg ctcactgagg
      781 ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc
      841 gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag ttaatgatta
      901 acccgccatg ctacttatct acgtagccat gctctaggaa gatctttccc atgattcctt
      961 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     1021 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     1081 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     1141 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggggcg aggagctgtt
     1201 caccggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     1261 aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag ttaaaataag
     1321 gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag aggtacccgt
     1381 tgatctaaca tatcctggtg tggagtaggg gacgctgctc tgacagaggc tcgggggcct
     1441 gagctggctc tgtgagctgg ggaggaggca gacagccagg ccttgtctgc aagcagacct
     1501 ggcagcattg ggctggccgc cccccagggc ctcctcttca tgcccagtga atgactcacc
     1561 ttggcacaga cacaatgttc ggggtgggca cagtgcctgc ttcccgccgc accccagccc
     1621 ccctcaaatg ccttccgaga agcccattga gcagggggct tgcattgcac cccagcctga
     1681 cagcctggca tcttgggata aaagcagcac agccccctag gggctgccct tgctgtgtgg
     1741 cgccaccggc ggtggagaac aaggctctat tcagcctgtg cccaggaaag gggatcaggg
     1801 gatgcccagg catggacagt gggtggcagg gggggagagg agggctgtct gcttcccaga
     1861 agtccaagga cacaaatggg tgaggggaga gctctcccca tagctgggct gcggcccaac
     1921 cccaccccct caggctatgc cagggggtgt tgccaggggc acccgggcat cgccagtcta
     1981 gcccactcct tcataaagcc ctcgcatccc aggagcgagc agagccagag caggttggag
     2041 aggagacgca tcacctccgc tgctcgcaag ctttattgcg gtagtttatc acagttaaat
     2101 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
     2161 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
     2221 actgggcttg tcgagacaga gaaaactctt gcgtttctga taggcaccta ttggtcttac
     2281 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     2341 aggctagagt acttaatacg actcactata ggctagcctc gagaattcac caccatggtg
     2401 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     2461 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     2521 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     2581 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     2641 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     2701 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     2761 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     2821 aagaaaacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     2881 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     2941 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     3001 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     3061 gagggccgcc actccaccgg cggcatggac gagctgtaca agtaactaga gtcgacccgg
     3121 gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     3181 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     3241 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     3301 tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
     3361 cgataaggat cttcctagag catggctacg tagataagta gcatggcggg ttaatcatta
     3421 actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     3481 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     3541 gcgagcgagc gcgcagcctt aattaaccta attcactggc cgtcgtttta caacgtcgtg
     3601 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     3661 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     3721 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3781 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3841 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3901 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3961 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4021 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4081 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4141 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     4201 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4261 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4321 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4381 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4441 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4501 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4561 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4621 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4681 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4741 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4801 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4861 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4921 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4981 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5041 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5101 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5161 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5221 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5281 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5341 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5401 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5461 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5521 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5581 agtggctgct gccagtggcg ataagtcgtg tcttaccggg tt
//
