LOCUS       sequence-495350-        4559 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tagging endogenously expressed proteins with ""spaghetti monster"" 
            smFP-V5, C-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4559)
  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 4559)
  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 smFP-V5 donor ORF2
FEATURES             Location/Qualifiers
     source          1..4559
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             833..874
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             884..925
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             935..976
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1508..1549
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1559..1600
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1619..1660
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1670..1711
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1925..1966
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1976..2017
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2027..2068
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_RNA        complement(2113..2188)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2209..2457)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2267..2286)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2437..2457)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2472..2612
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2472..2601
                     /label=AAV2 ITR
     rep_origin      2687..3142
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2774..2793)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2984..3005
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3159..3178)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3278..3300
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3338..3356)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3424..3528
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3529..3597,3598..4389)
                     /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(3747..3766)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgctc ggtccgcacg tggacgcttc cgagtacggt acaggctcga ggggaaaacc
      841 tataccgaac cccctccttg gacttgatag tactcagcag cagggtaagc ccattccaaa
      901 cccacttctc ggtctggata gcacacagca acaaggtaag cctatcccta accctctgtt
      961 gggcctggac tccacgggcc aaggtgtgag taagggcgaa gaactgttta ccggcgtggt
     1021 cccaattctg gttgagctcg acggtgacgt taacggacac aagtttagtg tgcgcggaga
     1081 aggagaggga gatgccacca acggaaaatt gacgctgaag tttatatgca ccacgggaaa
     1141 gctgccggtg ccgtggccga ccttggttac aaccctgggt ggcggagtcc agtgtttcag
     1201 ccgttaccca gaccacatga agcagcatga cttctttaag agtgctatgc cggagggata
     1261 cgtgcaggaa cggacgatca gtttcaaaga tgacggtact tacaagacgc gagccgaggt
     1321 gaagttcgag ggagacacct tggtgaacag gatagaactt aagggaattg atttcaagga
     1381 agacggtaat attctgggtc acaagctgga atacaacttt aacagccata acgtctacat
     1441 tactgccgac aagcagaaga acggcataaa ggcgaatttc aaaattcggc acaacgtgga
     1501 aggtggtggc aagccgatcc cgaatcccct gctgggcttg gatagcacgc agcaacaggg
     1561 aaagcccatc cctaacccct tgctgggact cgacagcacc ggaggccagc aaggcggtgg
     1621 caaaccgata cccaatcccc tgctcggcct ggatagcacc cagcagcaag gcaagccaat
     1681 ccccaaccct ctgctgggcc tcgactcgac aggtggcgat ggaagtgttc aactggcgga
     1741 ccactatcag cagaatacgc cgatcggaga tggccccgtg ctgttgccgg ataatcacta
     1801 tctgtcgact cagagcgttc tgagtaagga cccaaacgaa aaacgagatc acatggtgtt
     1861 gttggagttc gtgaccgccg ccggcattac gttgggaatg gacgagctgt acaagggcca
     1921 aggaggcaag cccataccaa atcccttgct gggcctcgac agcactcagc aacaaggaaa
     1981 gccaatacca aatccactgt tgggcctcga cagtacgcaa cagcagggca agcccatccc
     2041 gaacccgttg ctgggactgg atagtaccta aggatccgac gcttccgagt acggtacagg
     2101 tctagaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
     2161 tttaacttgc tatttctagc tctaaaacgt accgtactcg gaagcgtcgg tgtttcgtcc
     2221 tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg
     2281 atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct
     2341 acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc caattatctc
     2401 tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcacg
     2461 cgtgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     2521 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     2581 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     2641 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     2701 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2761 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2821 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2881 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     2941 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3001 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     3061 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     3121 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     3181 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3241 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3301 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     3361 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     3421 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3481 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     3541 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     3601 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     3661 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     3721 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     3781 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     3841 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     3901 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3961 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     4021 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     4081 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     4141 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     4201 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     4261 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     4321 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     4381 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     4441 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     4501 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
