LOCUS       sequence-430687-        6401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To tag Hevin with HA at the N-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6401)
  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 6401)
  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-N-HATag
FEATURES             Location/Qualifiers
     source          1..6401
                     /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..552
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     intron          1395..1527
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1510..1529
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1572..1590
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1611..1631
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3290..3411
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3301..3320)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3355..3374
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3573..3702
                     /label=AAV2 ITR
     primer_bind     complement(3721..3738)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3721..3737)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3730..3752)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3879..4334
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3966..3985)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4176..4197
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4351..4370)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4470..4492
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4530..4548)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4616..4720
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4721..4789,4790..5581)
                     /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      5752..6340
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6241..6260
                     /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 accgcgctac agtccccatt gccggtttta
      421 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      481 gagtcggtgc ttttttggat ccccccggca atggggactg tagcgtaccc ttacgacgtt
      541 ccagactacg ctaccccggc aatggggact gtagcggtcg actctagact gcagagggag
      601 atctaacata tcctggtgtg gagtagggga cgctgctctg acagaggctc gggggcctga
      661 gctggctctg tgagctgggg aggaggcaga cagccaggcc ttgtctgcaa gcagacctgg
      721 cagcattggg ctggccgccc cccagggcct cctcttcatg cccagtgaat gactcacctt
      781 ggcacagaca caatgttcgg ggtgggcaca gtgcctgctt cccgccgcac cccagccccc
      841 ctcaaatgcc ttccgagaag cccattgagc agggggcttg cattgcaccc cagcctgaca
      901 gcctggcatc ttgggataaa agcagcacag ccccctaggg gctgcccttg ctgtgtggcg
      961 ccaccggcgg tggagaacaa ggctctattc agcctgtgcc caggaaaggg gatcagggga
     1021 tgcccaggca tggacagtgg gtggcagggg gggagaggag ggctgtctgc ttcccagaag
     1081 tccaaggaca caaatgggtg aggggagagc tctccccata gctgggctgc ggcccaaccc
     1141 caccccctca ggctatgcca gggggtgttg ccaggggcac ccgggcatcg ccagtctagc
     1201 ccactccttc ataaagccct cgcatcccag gagcgagcag agccagagca ggttggagag
     1261 gagacgcatc acctccgctg ctcgcaagct ttattgcggt agtttatcac agttaaattg
     1321 ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcaga agttggtcgt
     1381 gaggcactgg gcaggtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac
     1441 tgggcttgtc gagacagaga agactcttgc gtttctgata ggcacctatt ggtcttactg
     1501 acatccactt tgcctttctc tccacaggtg tccactccca gttcaattac agctcttaag
     1561 gctagagtac ttaatacgac tcactatagg ctagcggtac cgccaccatg cccaagaaga
     1621 agaggaaggt gtccaatctc ctgactgttc accagaacct ccctgcgctg ccagtagatg
     1681 ccactagcga tgaggtcagg aaaaatctca tggatatgtt tagggataga caggcgtttt
     1741 ctgaacacac ctggaaaatg ctgcttagcg tgtgccgatc ctgggcagcc tggtgtaagc
     1801 tgaacaatcg caaatggttc cccgccgagc cggaggacgt gcgcgattac ctgctgtatc
     1861 tccaggcaag agggctggct gtcaagacta tccagcagca cttgggccaa ctgaatatgc
     1921 tgcatcgacg cagcgggctc ccccggccta gcgattcaaa cgcagtctcc cttgttatga
     1981 ggagaattag aaaggaaaac gtagatgcgg gtgagagggc taagcaggct ctcgcttttg
     2041 agcggactga tttcgaccag gtcagatccc tgatggagaa cagcgatcgg tgccaggaca
     2101 tcaggaacct cgcatttctg ggaattgcat ataacacact tctgcgcata gctgagatcg
     2161 cccggatcag agtgaaagac atcagtcgaa cggacggcgg ccggatgctt attcatattg
     2221 gacgcacaaa gacattggtc agcaccgctg gcgttgaaaa ggccttgtcc ctgggcgtaa
     2281 cgaagctggt ggaaagatgg atctcagtgt ccggcgtggc tgacgaccct aataattact
     2341 tgttctgtcg agtgagaaaa aacggagtcg ccgcgccctc tgccaccagc caattgagta
     2401 cacgggccct tgaagggatc tttgaggcaa cccaccgact catatacgga gccaaggatg
     2461 acagtggcca gaggtatctc gcctggtcag gtcattctgc tagggtgggg gccgcacgag
     2521 acatggcgcg ggcaggagtc tccataccag agattatgca agctggaggt tggacaaatg
     2581 tgaacatcgt tatgaactat atccgcaatc ttgactctga aaccggggcc atggtgagac
     2641 tgctcgaaga tggtgactag gaattcgata tctgctggaa gatggcgatt agcatatggg
     2701 cattcaccgc gtgccttact acgcgtattc ttaagcaata agcgaatata acacggtcga
     2761 tctaaaaagg ttagctgggt gtgttagaag ccctgcaaga cgacctgcgc attattactc
     2821 acggtacgac gcattattac tcacggtacg aggcattcac cgcgtgcctt actacgcgta
     2881 ttcttaagca ataagcgaat ataacacggt cgatctaaaa aggttagctg ggtgtgttag
     2941 aagccctgca agacgacctg ggcattcacc gcgtgcctta ctacgcgtat tcttaagcaa
     3001 taagcgaata taacacggtc gatctaaaaa ggttagctgg gtgtgttaga agccctgcaa
     3061 gacgacctgg gtaccgcatt attactcacg gtacgacgca ttattactca cggtacgagg
     3121 cattcaccgc gtgccttact acgcgtattc ttaagcaata agcgaatata acacggtcga
     3181 tctaaaaagg ttagctgggt gtgttagaag ccctgcaaga cgacctgggt accaggtaaa
     3241 gcggccgcga ctctgatatc aagcttgcgg ccgcttcgag cagacatgat aagatacatt
     3301 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     3361 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     3421 aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag
     3481 taaaacctct acaaatgtgg taaaatcgat aaggatcttc ctagagcatg gctacgtaga
     3541 taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac
     3601 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     3661 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agccttaatt aacctaattc
     3721 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     3781 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     3841 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     3901 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3961 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4021 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4081 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4141 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4201 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     4261 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4321 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4381 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4441 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4501 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     4561 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     4621 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4681 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4741 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4801 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4861 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4921 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     4981 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5041 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5101 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5161 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5221 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5281 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5341 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5401 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     5461 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5521 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5581 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     5641 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5701 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5761 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5821 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5881 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5941 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6001 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6061 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6121 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6181 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6241 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6301 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6361 tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
