LOCUS       sequence-232634-        4765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with Dronpa3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4765)
  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 4765)
  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-P16
FEATURES             Location/Qualifiers
     source          1..4765
                     /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"
     polyA_signal    1868..2092
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2099..2347
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2099..2119
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2270..2289
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2349..2382)
                     /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(2419..2438)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2420..2438)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2525..2542)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2696..3284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2776..2795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3455..4246,4247..4315))
                     /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     4078..4097
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4316..4420)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4488..4506
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4544..4566)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4666..4685
                     /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 gactgagtac aagcctactg tgcgtttagc caccagggat gatgtgccac gagccgtgag
     1321 gacactagca gccgcctttg ctgactaccc agctacaagg cacaccgtgg accctgacag
     1381 acacattgaa agagtcacag aacttcagga gctgttttta actagggtgg gcctggatat
     1441 tggcaaagtc tgggtggccg atgatggagc agcagttgca gtgtggacca ccccagagtc
     1501 tgttgaagct ggagctgtct ttgcagaaat tggaccaaga atggccgagt tatctggcag
     1561 caggctggct gctcaacagc agatggaagg attattagct ccccacagac ccaaggagcc
     1621 tgcctggttc ctggcaacag ttggagtttc tccagaccat cagggcaagg gcttaggatc
     1681 tgctgtggtg ctgcctggtg tggaagcagc tgagagagct ggggtgccag catttctgga
     1741 gacatcagct cctagaaacc tgcccttcta tgaacggctg ggcttcacag tgactgctga
     1801 tgtggaggtg cctgagggcc ccagaacctg gtgcatgaca agaaaaccag gagcctgact
     1861 gcagcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1921 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1981 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2041 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggctcgagga
     2101 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     2161 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     2221 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     2281 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     2341 aaacaccgat aacttcgtat agcatacatt atacgaagtt atggtaccga tgcagctagc
     2401 ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc caggttaacc
     2461 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2521 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2581 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2641 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2701 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2761 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2821 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2881 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2941 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3001 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3061 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3121 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3181 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3241 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3301 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3361 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3421 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3481 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3541 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3601 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3661 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3721 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3781 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3841 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3901 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3961 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4021 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4081 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4141 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4201 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4261 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4321 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4381 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4441 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4501 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     4561 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     4621 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     4681 ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca
     4741 taaccttatg tatcatacac atacg
//
