LOCUS       sequence-397520-        3391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tagging endogenously expressed proteins with HA epitope, C-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3391)
  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 3391)
  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 HA-epitope donor ORF2
FEATURES             Location/Qualifiers
     source          1..3391
                     /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..559
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(575..601)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     repeat_region   654..794
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   654..783
                     /label=AAV2 ITR
     rep_origin      869..1324
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(956..975)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1166..1187
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1341..1360)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1460..1482
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1520..1538)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1606..1710
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1711..1779,1780..2571)
                     /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(1929..1948)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2742..3330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3231..3250
                     /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 aataccctta
      541 cgatgtaccg gattacgcat agctagctag ctagagcata gtcaggaacg tcatagggat
      601 agctcgagcc tgtaccgtac tcggaagcgt ccacgtgcgg accgagcggc cgcaggaacc
      661 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
      721 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
      781 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
      841 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
      901 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
      961 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1021 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1081 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1141 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1201 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     1261 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     1321 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     1381 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     1441 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     1501 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     1561 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1621 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1681 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     1741 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     1801 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     1861 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1921 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     1981 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     2041 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     2101 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     2161 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     2221 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     2281 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     2341 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     2401 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     2461 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     2521 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     2581 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2641 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2701 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2761 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2821 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2881 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2941 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3001 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3061 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3121 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3181 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3241 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3301 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3361 ctggcctttt gctggccttt tgctcacatg t
//
