LOCUS       sequence-228134-        4540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with Dronpa3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4540)
  AUTHORS   Fueller J, Herbst K, Meurer M, Gubicza K, Kurtulmus B, Knopf JD, 
            Kirrmaier D, Buchmuller BC, Pereira G, Lemberg MK, Knop M
  TITLE     CRISPR-Cas12a-assisted PCR tagging of mammalian genes.
  JOURNAL   J Cell Biol. 2020 Jun 1;219(6). pii: 151766. doi: 
            10.1083/jcb.201910210.
  PUBMED    32406907
REFERENCE   2  (bases 1 to 4540)
  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"; journalName: "J Cell 
            Biol."; date: "2020-06-1"; volume: "219(6). pii: 151766. doi"; 
            pages: " 10.1083/jcb.201910210"
COMMENT     Sequence Label: pMaCTag-Z16
FEATURES             Location/Qualifiers
     source          1..4540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..738
                     /codon_start=1
                     /product="photoswitchable Dronpa-Green3 fluorescent 
                     protein"
                     /label=Dronpa-Green3
                     /translation="VSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVK
                     EGGPLPFAYDILTTVFCYGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNAT
                     NDITLDGDCYIYEIRFDGVNFPANGPVMQKRTVKWEPSTEKLYVRDGVLKGDINAALSL
                     EGGGHYRCDFKTTYKAKKVVQLPDYHFVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQ
                     AK"
     polyA_signal    745..866
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(782..801)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     836..855
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(873..906)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        913..1229
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1080..1215
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1236..1245
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1242..1616
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1626..1850
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1857..2105
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1857..1877
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2028..2047
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2107..2140)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2177..2196)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2178..2196)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2283..2300)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2454..3042)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2534..2553)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3213..4004,4005..4073))
                     /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     3836..3855
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4074..4178)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4246..4264
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4302..4324)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4424..4443
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4524..4540,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4524..4540,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagtg tgattaaacc agacatgaag atcaagctgc gtatggaagg cgctgtaaat
      121 ggacacccgt tcgcgattga aggagttggc cttgggaagc ctttcgaggg aaaacagagt
      181 atggacctta aagtcaaaga aggcggacct ctgcctttcg cctatgacat cttgacaact
      241 gtgttctgtt acggcaacag ggtattcgcc aaatacccag aaaatatagt agactatttc
      301 aagcagtcgt ttcctgaggg ctactcttgg gaacgaagca tgaattacga agacgggggc
      361 atttgtaacg cgacaaacga cataaccctg gatggtgact gttatatcta tgaaattcga
      421 tttgatggtg tgaactttcc tgccaatggt ccagttatgc agaagaggac tgtgaaatgg
      481 gagccatcca ctgagaaatt gtatgtgcgt gatggagtgc tgaagggtga tattaacgcg
      541 gctctgtcgc ttgaaggagg tggccattac cgatgtgact tcaaaactac ttataaagct
      601 aagaaggttg tccagttgcc agactatcac tttgtggacc accacattga gattaaaagc
      661 cacgacaaag attacagtaa tgttaatctg catgagcacg ccgaagcgca ttctgagctg
      721 ccgaggcagg ccaagtgaac tagtaacttg tttattgcag cttataatgg ttacaaataa
      781 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
      841 ttgtccaaac tcatcaatgt atcttagtcg atataacttc gtatagcata cattatacga
      901 agttatgtcg acggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
      961 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     1021 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     1081 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     1141 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     1201 gcttttttgg aggcctaggc ttttgcaaag aattcgccac catggccaag ttgaccagtg
     1261 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     1321 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     1381 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     1441 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     1501 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     1561 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgactgc
     1621 agcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     1681 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     1741 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     1801 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg ctcgaggagg
     1861 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     1921 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     1981 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     2041 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     2101 acaccgataa cttcgtatag catacattat acgaagttat ggtaccgatg cagctagccc
     2161 gcggatctgc cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg
     2221 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2281 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2341 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2401 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2461 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2521 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2581 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2641 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2701 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2761 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2821 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2881 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2941 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3001 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3061 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3121 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3181 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3241 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3301 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3361 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3421 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3481 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3541 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3601 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3661 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3721 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3781 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3841 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3901 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3961 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4021 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4081 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4141 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4201 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     4261 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     4321 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     4381 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     4441 gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata
     4501 accttatgta tcatacacat acgatttagg tgacactata
//
