LOCUS       sequence-430691-        7242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To KO and genetrap mouse Hevin simultaneously.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7242)
  AUTHORS   Bindu DS, Tan CX, Savage JT, Eroglu C
  TITLE     GEARBOCS: An Adeno Associated Virus Tool for In Vivo Gene Editing in
            Astrocytes.
  JOURNAL   bioRxiv. 2023 Jan 19:2023.01.17.524433. doi: 
            10.1101/2023.01.17.524433. Preprint.
  PUBMED    36711516
REFERENCE   2  (bases 1 to 7242)
  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: "bioRxiv. 
            2023 Jan 19:2023.01.17.524433. doi: 10.1101/2023.01.17.524433. 
            Preprint."
COMMENT     Sequence Label: GEARBOCS-SPARCL1-GeneTRAP
FEATURES             Location/Qualifiers
     source          1..7242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        415..490
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             526..1230
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(668..686)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     936..955
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1272..1393
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     intron          2236..2368
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2351..2370
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        2413..2431
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2452..2472
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4131..4252
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   4414..4543
                     /label=AAV2 ITR
     primer_bind     complement(4562..4579)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4562..4578)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4571..4593)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4720..5175
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4807..4826)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5017..5038
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5192..5211)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5311..5333
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5371..5389)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5457..5561
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5562..5630,5631..6422)
                     /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      6593..7181
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7082..7101
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accggtgcgc cttgggaacc gctggtttta
      421 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      481 gagtcggtgc ttttttggat ccccacagcg gttcccaagg cgcacgtgag caagggcgag
      541 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
      601 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
      661 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
      721 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
      781 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
      841 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
      901 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
      961 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     1021 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     1081 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     1141 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     1201 tccaccggcg gcatggacga gctgtacaag tgctgcatca tcctgtaagc ggccgcttcg
     1261 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     1321 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     1381 caataaacaa gttaccacag cggttcccaa ggcgcacgtc gactctagac tgcagaggga
     1441 gatctaacat atcctggtgt ggagtagggg acgctgctct gacagaggct cgggggcctg
     1501 agctggctct gtgagctggg gaggaggcag acagccaggc cttgtctgca agcagacctg
     1561 gcagcattgg gctggccgcc ccccagggcc tcctcttcat gcccagtgaa tgactcacct
     1621 tggcacagac acaatgttcg gggtgggcac agtgcctgct tcccgccgca ccccagcccc
     1681 cctcaaatgc cttccgagaa gcccattgag cagggggctt gcattgcacc ccagcctgac
     1741 agcctggcat cttgggataa aagcagcaca gccccctagg ggctgccctt gctgtgtggc
     1801 gccaccggcg gtggagaaca aggctctatt cagcctgtgc ccaggaaagg ggatcagggg
     1861 atgcccaggc atggacagtg ggtggcaggg ggggagagga gggctgtctg cttcccagaa
     1921 gtccaaggac acaaatgggt gaggggagag ctctccccat agctgggctg cggcccaacc
     1981 ccaccccctc aggctatgcc agggggtgtt gccaggggca cccgggcatc gccagtctag
     2041 cccactcctt cataaagccc tcgcatccca ggagcgagca gagccagagc aggttggaga
     2101 ggagacgcat cacctccgct gctcgcaagc tttattgcgg tagtttatca cagttaaatt
     2161 gctaacgcag tcagtgcttc tgacacaaca gtctcgaact taagctgcag aagttggtcg
     2221 tgaggcactg ggcaggtaag tatcaaggtt acaagacagg tttaaggaga ccaatagaaa
     2281 ctgggcttgt cgagacagag aagactcttg cgtttctgat aggcacctat tggtcttact
     2341 gacatccact ttgcctttct ctccacaggt gtccactccc agttcaatta cagctcttaa
     2401 ggctagagta cttaatacga ctcactatag gctagcggta ccgccaccat gcccaagaag
     2461 aagaggaagg tgtccaatct cctgactgtt caccagaacc tccctgcgct gccagtagat
     2521 gccactagcg atgaggtcag gaaaaatctc atggatatgt ttagggatag acaggcgttt
     2581 tctgaacaca cctggaaaat gctgcttagc gtgtgccgat cctgggcagc ctggtgtaag
     2641 ctgaacaatc gcaaatggtt ccccgccgag ccggaggacg tgcgcgatta cctgctgtat
     2701 ctccaggcaa gagggctggc tgtcaagact atccagcagc acttgggcca actgaatatg
     2761 ctgcatcgac gcagcgggct cccccggcct agcgattcaa acgcagtctc ccttgttatg
     2821 aggagaatta gaaaggaaaa cgtagatgcg ggtgagaggg ctaagcaggc tctcgctttt
     2881 gagcggactg atttcgacca ggtcagatcc ctgatggaga acagcgatcg gtgccaggac
     2941 atcaggaacc tcgcatttct gggaattgca tataacacac ttctgcgcat agctgagatc
     3001 gcccggatca gagtgaaaga catcagtcga acggacggcg gccggatgct tattcatatt
     3061 ggacgcacaa agacattggt cagcaccgct ggcgttgaaa aggccttgtc cctgggcgta
     3121 acgaagctgg tggaaagatg gatctcagtg tccggcgtgg ctgacgaccc taataattac
     3181 ttgttctgtc gagtgagaaa aaacggagtc gccgcgccct ctgccaccag ccaattgagt
     3241 acacgggccc ttgaagggat ctttgaggca acccaccgac tcatatacgg agccaaggat
     3301 gacagtggcc agaggtatct cgcctggtca ggtcattctg ctagggtggg ggccgcacga
     3361 gacatggcgc gggcaggagt ctccatacca gagattatgc aagctggagg ttggacaaat
     3421 gtgaacatcg ttatgaacta tatccgcaat cttgactctg aaaccggggc catggtgaga
     3481 ctgctcgaag atggtgacta ggaattcgat atctgctgga agatggcgat tagcatatgg
     3541 gcattcaccg cgtgccttac tacgcgtatt cttaagcaat aagcgaatat aacacggtcg
     3601 atctaaaaag gttagctggg tgtgttagaa gccctgcaag acgacctgcg cattattact
     3661 cacggtacga cgcattatta ctcacggtac gaggcattca ccgcgtgcct tactacgcgt
     3721 attcttaagc aataagcgaa tataacacgg tcgatctaaa aaggttagct gggtgtgtta
     3781 gaagccctgc aagacgacct gggcattcac cgcgtgcctt actacgcgta ttcttaagca
     3841 ataagcgaat ataacacggt cgatctaaaa aggttagctg ggtgtgttag aagccctgca
     3901 agacgacctg ggtaccgcat tattactcac ggtacgacgc attattactc acggtacgag
     3961 gcattcaccg cgtgccttac tacgcgtatt cttaagcaat aagcgaatat aacacggtcg
     4021 atctaaaaag gttagctggg tgtgttagaa gccctgcaag acgacctggg taccaggtaa
     4081 agcggccgcg actctgatat caagcttgcg gccgcttcga gcagacatga taagatacat
     4141 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     4201 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     4261 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa
     4321 gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt cctagagcat ggctacgtag
     4381 ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca
     4441 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     4501 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat taacctaatt
     4561 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     4621 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     4681 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     4741 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4801 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4861 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4921 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4981 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     5041 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     5101 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5161 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5221 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5281 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5341 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     5401 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     5461 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5521 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5581 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5641 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5701 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5761 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5821 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5881 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5941 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     6001 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6061 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6121 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6181 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6241 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6301 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6361 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6421 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6481 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6541 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6601 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6661 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6721 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6781 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6841 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6901 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6961 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7021 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7081 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7141 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7201 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
