LOCUS       sequence-397388-        4558 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 4558)
  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 4558)
  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 ORF0
FEATURES             Location/Qualifiers
     source          1..4558
                     /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             complement(534..575)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(585..626)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(636..677)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(891..932)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(942..983)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1002..1043)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1053..1094)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1626..1667)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1677..1718)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1728..1769)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     repeat_region   1821..1961
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1821..1950
                     /label=AAV2 ITR
     rep_origin      2036..2491
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2123..2142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2333..2354
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2508..2527)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2627..2649
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2687..2705)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2773..2877
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2878..2946,2947..3738)
                     /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(3096..3115)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3909..4497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4398..4417
                     /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 ttaggtacta
      541 tccagtccca gcaacgggtt cgggatgggc ttgccctgct gttgcgtact gtcgaggccc
      601 aacagtggat ttggtattgg ctttccttgt tgctgagtgc tgtcgaggcc cagcaaggga
      661 tttggtatgg gcttgcctcc ttggcccttg tacagctcgt ccattcccaa cgtaatgccg
      721 gcggcggtca cgaactccaa caacaccatg tgatctcgtt tttcgtttgg gtccttactc
      781 agaacgctct gagtcgacag atagtgatta tccggcaaca gcacggggcc atctccgatc
      841 ggcgtattct gctgatagtg gtccgccagt tgaacacttc catcgccacc tgtcgagtcg
      901 aggcccagca gagggttggg gattggcttg ccttgctgct gggtgctatc caggccgagc
      961 aggggattgg gtatcggttt gccaccgcct tgctggcctc cggtgctgtc gagtcccagc
     1021 aaggggttag ggatgggctt tccctgttgc tgcgtgctat ccaagcccag caggggattc
     1081 gggatcggct tgccaccacc ttccacgttg tgccgaattt tgaaattcgc ctttatgccg
     1141 ttcttctgct tgtcggcagt aatgtagacg ttatggctgt taaagttgta ttccagcttg
     1201 tgacccagaa tattaccgtc ttccttgaaa tcaattccct taagttctat cctgttcacc
     1261 aaggtgtctc cctcgaactt cacctcggct cgcgtcttgt aagtaccgtc atctttgaaa
     1321 ctgatcgtcc gttcctgcac gtatccctcc ggcatagcac tcttaaagaa gtcatgctgc
     1381 ttcatgtggt ctgggtaacg gctgaaacac tggactccgc cacccagggt tgtaaccaag
     1441 gtcggccacg gcaccggcag ctttcccgtg gtgcatataa acttcagcgt caattttccg
     1501 ttggtggcat ctccctctcc ttctccgcgc acactaaact tgtgtccgtt aacgtcaccg
     1561 tcgagctcaa ccagaattgg gaccacgccg gtaaacagtt cttcgccctt actcacacct
     1621 tggcccgtgg agtccaggcc caacagaggg ttagggatag gcttaccttg ttgctgtgtg
     1681 ctatccagac cgagaagtgg gtttggaatg ggcttaccct gctgctgagt actatcaagt
     1741 ccaaggaggg ggttcggtat aggttttccc tcgagcctgt accgtactcg gaagcgtcca
     1801 cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     1861 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     1921 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     1981 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     2041 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2101 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2161 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2221 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     2281 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2341 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     2401 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2461 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     2521 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     2581 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     2641 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     2701 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     2761 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2821 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2881 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2941 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3001 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3061 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3121 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3181 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3241 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3301 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3361 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3421 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3481 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3541 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3601 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3661 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3721 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3781 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4021 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
