LOCUS       sequence-232668-        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"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             81..755
                     /codon_start=1
                     /product="photoswitchable Dronpa-Green3 fluorescent 
                     protein"
                     /label=Dronpa-Green3
                     /translation="VSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVK
                     EGGPLPFAYDILTTVFCYGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNAT
                     NDITLDGDCYIYEIRFDGVNFPANGPVMQKRTVKWEPSTEKLYVRDGVLKGDINAALSL
                     EGGGHYRCDFKTTYKAKKVVQLPDYHFVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQ
                     AK"
     polyA_signal    762..883
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(799..818)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     853..872
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(890..923)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        930..1246
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1097..1232
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1253..1262
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1259..1633
                     /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    1643..1867
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1874..2122
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1874..1894
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2045..2064
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2124..2157)
                     /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(2194..2213)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2195..2213)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2300..2317)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2471..3059)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2551..2570)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3230..4021,4022..4090))
                     /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     3853..3872
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4091..4195)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4263..4281
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4319..4341)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4441..4460
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagtgtga ttaaaccaga catgaagatc aagctgcgta
      121 tggaaggcgc tgtaaatgga cacccgttcg cgattgaagg agttggcctt gggaagcctt
      181 tcgagggaaa acagagtatg gaccttaaag tcaaagaagg cggacctctg cctttcgcct
      241 atgacatctt gacaactgtg ttctgttacg gcaacagggt attcgccaaa tacccagaaa
      301 atatagtaga ctatttcaag cagtcgtttc ctgagggcta ctcttgggaa cgaagcatga
      361 attacgaaga cgggggcatt tgtaacgcga caaacgacat aaccctggat ggtgactgtt
      421 atatctatga aattcgattt gatggtgtga actttcctgc caatggtcca gttatgcaga
      481 agaggactgt gaaatgggag ccatccactg agaaattgta tgtgcgtgat ggagtgctga
      541 agggtgatat taacgcggct ctgtcgcttg aaggaggtgg ccattaccga tgtgacttca
      601 aaactactta taaagctaag aaggttgtcc agttgccaga ctatcacttt gtggaccacc
      661 acattgagat taaaagccac gacaaagatt acagtaatgt taatctgcat gagcacgccg
      721 aagcgcattc tgagctgccg aggcaggcca agtgaactag taacttgttt attgcagctt
      781 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
      841 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttagtcgata taacttcgta
      901 tagcatacat tatacgaagt tatgtcgacg gtgtggaaag tccccaggct ccccagcagg
      961 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     1021 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     1081 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     1141 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     1201 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagaat tcgccaccat
     1261 ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga
     1321 gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt
     1381 ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa
     1441 caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt
     1501 cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc
     1561 gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga
     1621 ggagcaggac tgactgcagc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     1681 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     1741 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     1801 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     1861 tctatggctc gaggagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     1921 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     1981 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2041 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2101 cttgtggaaa ggacgaaaca ccgataactt cgtatagcat acattatacg aagttatggt
     2161 accgatgcag ctagcccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga
     2221 taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2281 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2341 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2401 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2461 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2521 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2581 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2641 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2701 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2761 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2821 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2881 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     2941 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3001 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3061 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3121 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3181 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3241 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3301 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3361 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3421 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3481 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3541 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3601 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3661 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3721 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3781 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3841 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3901 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3961 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4021 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4081 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4141 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4201 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     4261 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     4321 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     4381 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
     4441 cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg
     4501 gctacaatta atacataacc ttatgtatca tacacatacg
//
