LOCUS       sequence-232517-        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"
     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..1178
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     930..950
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1101..1120
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1180..1213)
                     /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(1250..1269)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1251..1269)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1356..1373)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1527..2115)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1607..1626)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2286..3077,3078..3146))
                     /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     2909..2928
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3147..3251)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3319..3337
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3375..3397)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3497..3516
                     /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 tatgtcgagg agggcctatt tcccatgatt ccttcatatt
      961 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     1021 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     1081 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     1141 tcttggcttt atatatcttg tggaaaggac gaaacaccga taacttcgta tagcatacat
     1201 tatacgaagt tatggtaccg atgcagctag cccgcggatc tgccggtctc cctatagtga
     1261 gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg
     1321 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     1381 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     1441 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1501 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1561 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1621 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1681 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1741 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1801 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1861 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1921 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     1981 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2041 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2101 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2161 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     2221 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2281 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     2341 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     2401 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     2461 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     2521 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     2581 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     2641 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     2701 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     2761 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     2821 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     2881 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     2941 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3001 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3061 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3121 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     3181 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3241 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     3301 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     3361 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     3421 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     3481 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatggacata
     3541 ttgtcgttag aacgcggcta caattaatac ataaccttat gtatcataca catacg
//
