LOCUS       sequence-228019-        3596 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with Dronpa3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3596)
  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 3596)
  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-16
FEATURES             Location/Qualifiers
     source          1..3596
                     /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..1161
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     913..933
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1084..1103
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1163..1196)
                     /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(1233..1252)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1234..1252)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1339..1356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1510..2098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1590..1609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2269..3060,3061..3129))
                     /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     2892..2911
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3130..3234)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3302..3320
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3358..3380)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3480..3499
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3580..3596,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3580..3596,1)
                     /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 aggagggcct atttcccatg attccttcat atttgcatat acgatacaag
      961 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     1021 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     1081 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     1141 ttgtggaaag gacgaaacac cgataacttc gtatagcata cattatacga agttatggta
     1201 ccgatgcagc tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat
     1261 aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1321 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1381 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     1441 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     1501 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1561 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1621 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1681 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
     1741 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     1801 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1861 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     1921 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     1981 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2041 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2101 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     2161 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     2221 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     2281 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     2341 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     2401 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     2461 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     2521 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     2581 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     2641 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     2701 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     2761 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     2821 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     2881 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     2941 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     3001 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     3061 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     3121 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     3181 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     3241 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     3301 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     3361 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     3421 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     3481 ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg
     3541 ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac actata
//
