LOCUS       sequence-228062-        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"
     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"
     polyA_signal    1851..2075
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2082..2330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2082..2102
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2253..2272
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2332..2365)
                     /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(2402..2421)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2403..2421)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2508..2525)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2679..3267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2759..2778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3438..4229,4230..4298))
                     /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     4061..4080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4299..4403)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4471..4489
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4527..4549)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4649..4668
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4749..4765,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4749..4765,1)
                     /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 catgactgag tacaagccta
     1261 ctgtgcgttt agccaccagg gatgatgtgc cacgagccgt gaggacacta gcagccgcct
     1321 ttgctgacta cccagctaca aggcacaccg tggaccctga cagacacatt gaaagagtca
     1381 cagaacttca ggagctgttt ttaactaggg tgggcctgga tattggcaaa gtctgggtgg
     1441 ccgatgatgg agcagcagtt gcagtgtgga ccaccccaga gtctgttgaa gctggagctg
     1501 tctttgcaga aattggacca agaatggccg agttatctgg cagcaggctg gctgctcaac
     1561 agcagatgga aggattatta gctccccaca gacccaagga gcctgcctgg ttcctggcaa
     1621 cagttggagt ttctccagac catcagggca agggcttagg atctgctgtg gtgctgcctg
     1681 gtgtggaagc agctgagaga gctggggtgc cagcatttct ggagacatca gctcctagaa
     1741 acctgccctt ctatgaacgg ctgggcttca cagtgactgc tgatgtggag gtgcctgagg
     1801 gccccagaac ctggtgcatg acaagaaaac caggagcctg actgcagcga ctgtgccttc
     1861 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     1921 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     1981 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2041 tagcaggcat gctggggatg cggtgggctc tatggctcga ggagggccta tttcccatga
     2101 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     2161 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     2221 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     2281 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gataacttcg
     2341 tatagcatac attatacgaa gttatggtac cgatgcagct agcccgcgga tctgccggtc
     2401 tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc
     2461 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2521 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2581 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2641 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2701 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2761 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2821 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     2881 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2941 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3001 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3061 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     3121 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3181 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3241 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3301 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3361 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3421 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3481 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3541 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3601 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3661 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3721 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3781 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3841 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3901 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3961 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4021 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4081 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4141 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4201 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4261 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4321 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4381 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     4441 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     4501 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     4561 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     4621 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     4681 catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata
     4741 cacatacgat ttaggtgaca ctata
//
