LOCUS       sequence-430694-        7079 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To tag Hevin with mCherry at its C-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7079)
  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 7079)
  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-C-mCherryTag
FEATURES             Location/Qualifiers
     source          1..7079
                     /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"
     intron          2073..2205
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2188..2207
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        2250..2268
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2289..2309
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3968..4089
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3979..3998)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4033..4052
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4251..4380
                     /label=AAV2 ITR
     primer_bind     complement(4399..4416)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4399..4415)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4408..4430)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4557..5012
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4644..4663)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4854..4875
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5029..5048)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5148..5170
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5208..5226)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5294..5398
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5399..5467,5468..6259)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLQNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6430..7018
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6919..6938
                     /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 accgcgagtt atgcagtgtt ccatgtttta
      421 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      481 gagtcggtgc ttttttggat cccgagttat gcagtgttcc atggggtgag 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 tcgagttatg cagtgttcca tggggtcgac
     1261 tctagactgc agagggagat ctaacatatc ctggtgtgga gtaggggacg ctgctctgac
     1321 agaggctcgg gggcctgagc tggctctgtg agctggggag gaggcagaca gccaggcctt
     1381 gtctgcaagc agacctggca gcattgggct ggccgccccc cagggcctcc tcttcatgcc
     1441 cagtgaatga ctcaccttgg cacagacaca atgttcgggg tgggcacagt gcctgcttcc
     1501 cgccgcaccc cagcccccct caaatgcctt ccgagaagcc cattgagcag ggggcttgca
     1561 ttgcacccca gcctgacagc ctggcatctt gggataaaag cagcacagcc ccctaggggc
     1621 tgcccttgct gtgtggcgcc accggcggtg gagaacaagg ctctattcag cctgtgccca
     1681 ggaaagggga tcaggggatg cccaggcatg gacagtgggt ggcagggggg gagaggaggg
     1741 ctgtctgctt cccagaagtc caaggacaca aatgggtgag gggagagctc tccccatagc
     1801 tgggctgcgg cccaacccca ccccctcagg ctatgccagg gggtgttgcc aggggcaccc
     1861 gggcatcgcc agtctagccc actccttcat aaagccctcg catcccagga gcgagcagag
     1921 ccagagcagg ttggagagga gacgcatcac ctccgctgct cgcaagcttt attgcggtag
     1981 tttatcacag ttaaattgct aacgcagtca gtgcttctga cacaacagtc tcgaacttaa
     2041 gctgcagaag ttggtcgtga ggcactgggc aggtaagtat caaggttaca agacaggttt
     2101 aaggagacca atagaaactg ggcttgtcga gacagagaag actcttgcgt ttctgatagg
     2161 cacctattgg tcttactgac atccactttg cctttctctc cacaggtgtc cactcccagt
     2221 tcaattacag ctcttaaggc tagagtactt aatacgactc actataggct agcggtaccg
     2281 ccaccatgcc caagaagaag aggaaggtgt ccaatctcct gactgttcac cagaacctcc
     2341 ctgcgctgcc agtagatgcc actagcgatg aggtcaggaa aaatctcatg gatatgttta
     2401 gggatagaca ggcgttttct gaacacacct ggaaaatgct gcttagcgtg tgccgatcct
     2461 gggcagcctg gtgtaagctg aacaatcgca aatggttccc cgccgagccg gaggacgtgc
     2521 gcgattacct gctgtatctc caggcaagag ggctggctgt caagactatc cagcagcact
     2581 tgggccaact gaatatgctg catcgacgca gcgggctccc ccggcctagc gattcaaacg
     2641 cagtctccct tgttatgagg agaattagaa aggaaaacgt agatgcgggt gagagggcta
     2701 agcaggctct cgcttttgag cggactgatt tcgaccaggt cagatccctg atggagaaca
     2761 gcgatcggtg ccaggacatc aggaacctcg catttctggg aattgcatat aacacacttc
     2821 tgcgcatagc tgagatcgcc cggatcagag tgaaagacat cagtcgaacg gacggcggcc
     2881 ggatgcttat tcatattgga cgcacaaaga cattggtcag caccgctggc gttgaaaagg
     2941 ccttgtccct gggcgtaacg aagctggtgg aaagatggat ctcagtgtcc ggcgtggctg
     3001 acgaccctaa taattacttg ttctgtcgag tgagaaaaaa cggagtcgcc gcgccctctg
     3061 ccaccagcca attgagtaca cgggcccttg aagggatctt tgaggcaacc caccgactca
     3121 tatacggagc caaggatgac agtggccaga ggtatctcgc ctggtcaggt cattctgcta
     3181 gggtgggggc cgcacgagac atggcgcggg caggagtctc cataccagag attatgcaag
     3241 ctggaggttg gacaaatgtg aacatcgtta tgaactatat ccgcaatctt gactctgaaa
     3301 ccggggccat ggtgagactg ctcgaagatg gtgactagga attcgatatc tgctggaaga
     3361 tggcgattag catatgggca ttcaccgcgt gccttactac gcgtattctt aagcaataag
     3421 cgaatataac acggtcgatc taaaaaggtt agctgggtgt gttagaagcc ctgcaagacg
     3481 acctgcgcat tattactcac ggtacgacgc attattactc acggtacgag gcattcaccg
     3541 cgtgccttac tacgcgtatt cttaagcaat aagcgaatat aacacggtcg atctaaaaag
     3601 gttagctggg tgtgttagaa gccctgcaag acgacctggg cattcaccgc gtgccttact
     3661 acgcgtattc ttaagcaata agcgaatata acacggtcga tctaaaaagg ttagctgggt
     3721 gtgttagaag ccctgcaaga cgacctgggt accgcattat tactcacggt acgacgcatt
     3781 attactcacg gtacgaggca ttcaccgcgt gccttactac gcgtattctt aagcaataag
     3841 cgaatataac acggtcgatc taaaaaggtt agctgggtgt gttagaagcc ctgcaagacg
     3901 acctgggtac caggtaaagc ggccgcgact ctgatatcaa gcttgcggcc gcttcgagca
     3961 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     4021 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     4081 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg
     4141 gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatcttcct
     4201 agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct
     4261 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4321 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4381 ccttaattaa cctaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     4441 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     4501 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     4561 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4621 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4681 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4741 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4801 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4861 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     4921 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4981 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     5041 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     5101 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5161 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     5221 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5281 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5341 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5401 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5461 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5521 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5581 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5641 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5701 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5761 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5821 aggaccgaag gagctaaccg cttttttgca aaacatgggg gatcatgtaa ctcgccttga
     5881 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5941 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6001 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6061 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6121 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6181 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6241 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6301 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6361 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6421 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6481 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6541 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6601 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6661 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6721 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6781 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6841 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6901 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6961 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7021 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
