LOCUS       sequence-405503-        3421 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 3421)
  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 3421)
  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 ORF2
FEATURES             Location/Qualifiers
     source          1..3421
                     /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             533..574
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(590..631)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     repeat_region   684..824
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   684..813
                     /label=AAV2 ITR
     rep_origin      899..1354
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(986..1005)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1196..1217
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1371..1390)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1490..1512
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1550..1568)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1636..1740
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1741..1809,1810..2601)
                     /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(1959..1978)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2772..3360
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3261..3280
                     /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 aaggtaagcc
      541 tatccctaac cctctcctcg gtctcgattc tacgtagcta gctagctaga gtggagtcca
      601 gtcccagaag tggattggga atgggtttgc cgctcgagcc tgtaccgtac tcggaagcgt
      661 ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
      721 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
      781 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
      841 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
      901 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
      961 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1021 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1081 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     1141 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1201 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     1261 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1321 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     1381 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     1441 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     1501 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     1561 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     1621 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1681 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1741 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1801 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     1861 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     1921 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     1981 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     2041 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     2101 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     2161 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     2221 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2281 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2341 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2401 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     2461 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     2521 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2581 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     2641 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     2701 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2761 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2821 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2881 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     2941 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3001 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3061 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3121 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3181 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3241 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3301 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3361 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3421 t
//
