LOCUS       sequence-397389-        4560 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 4560)
  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 4560)
  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 ORF1
FEATURES             Location/Qualifiers
     source          1..4560
                     /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   1823..1963
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1823..1952
                     /label=AAV2 ITR
     rep_origin      2038..2493
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2125..2144)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2335..2356
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2510..2529)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2629..2651
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2689..2707)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2775..2879
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2880..2948,2949..3740)
                     /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(3098..3117)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3911..4499
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4400..4419
                     /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 tctcgagcct gtaccgtact cggaagcgtc
     1801 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     1861 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     1921 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     1981 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     2041 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2101 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2161 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2221 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     2281 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2341 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     2401 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2461 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     2521 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     2581 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     2641 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     2701 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     2761 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2821 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2881 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2941 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3001 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3061 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3121 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3181 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3241 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3301 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     3361 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     3421 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3481 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3541 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     3601 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3661 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3721 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3781 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3841 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3901 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3961 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4021 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4081 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4141 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4201 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4261 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4321 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4381 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4441 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4501 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
