LOCUS       sequence-397531-        3422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tagging endogenously expressed proteins with V5 epitope, C-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3422)
  AUTHORS   Gao Y, Hisey E, Bradshaw TWA, Erata E, Brown WE, Courtland JL, Uezu 
            A, Xiang Y, Diao Y, Soderling SH
  TITLE     Plug-and-Play Protein Modification Using Homology-Independent 
            Universal Genome Engineering.
  JOURNAL   Neuron. 2019 Aug 21;103(4):583-597.e8. doi: 
            10.1016/j.neuron.2019.05.047. Epub 2019 Jul 1.
  PUBMED    31272828
REFERENCE   2  (bases 1 to 3422)
  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: 
            "10.1016/j.neuron.2019.05.047"; journalName: "Neuron"; date: 
            "2019-08-21- 21"; volume: "103"; issue: "4"; pages: "583-597.e8"
COMMENT     Sequence Label: pHiUGE V5-epitope donor ORF0
FEATURES             Location/Qualifiers
     source          1..3422
                     /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        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             532..573
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(589..630)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     repeat_region   685..825
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   685..814
                     /label=AAV2 ITR
     rep_origin      900..1355
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(987..1006)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1197..1218
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1372..1391)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1491..1513
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1551..1569)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1637..1741
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1742..1810,1811..2602)
                     /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"
     primer_bind     complement(1960..1979)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2773..3361
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3262..3281
                     /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 accgacgctt ccgagtacgg tacgttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag acctgtaccg tactcggaag cgtcggatcc aggtaagcct
      541 atccctaacc ctctcctcgg tctcgattct acgtagctag ctagctagag tggagtccag
      601 tcccagaagt ggattgggaa tgggtttgcc aagctcgagc ctgtaccgta ctcggaagcg
      661 tccacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
      721 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
      781 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
      841 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
      901 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
      961 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1021 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1081 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     1141 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1201 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     1261 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1321 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     1381 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     1441 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     1501 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     1561 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     1621 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1681 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     1741 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     1801 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     1861 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     1921 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     1981 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2041 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2101 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2161 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2221 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2281 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2341 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2401 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     2461 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2521 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2581 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     2641 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     2701 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2761 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2821 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2881 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2941 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3001 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3061 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3121 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3181 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3241 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3301 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3361 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3421 gt
//
