LOCUS       sequence-228053-        4786 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mScarlet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4786)
  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 4786)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-P12
FEATURES             Location/Qualifiers
     source          1..4786
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..756
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(194..212)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     462..481
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    766..887
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(803..822)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     857..876
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(894..927)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        934..1250
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1101..1236
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1872..2096
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2103..2351
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2103..2123
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2274..2293
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2353..2386)
                     /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(2423..2442)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2424..2442)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2529..2546)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2700..3288)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2780..2799)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3459..4250,4251..4319))
                     /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     4082..4101
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4320..4424)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4492..4510
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4548..4570)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4670..4689
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4770..4786,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4770..4786,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgaggc agtgatcaag gagttcatgc ggttcaaggt gcacatggag
      121 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
      181 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt ctcctgggac
      241 atcctgtccc ctcagttcat gtacggctcc agggccttca ccaagcaccc cgccgacatc
      301 cccgactact ataagcagtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
      361 gaggacggcg gcgccgtgac cgtgacccag gacacctccc tggaggacgg caccctgatc
      421 tacaaggtga agctccgcgg caccaacttc cctcctgacg gccccgtaat gcagaagaag
      481 acaatgggct gggaagcgtc caccgagcgg ttgtaccccg aggacggcgt gctgaagggc
      541 gacattaaga tggccctgcg cctgaaggac ggcggccgct acctggcgga cttcaagacc
      601 acctacaagg ccaagaagcc cgtgcagatg cccggcgcct acaacgtcga ccgcaagttg
      661 gacatcacct cccacaacga ggactacacc gtggtggaac agtacgaacg ctccgagggc
      721 cgccactcca ccggcggcat ggacgagctg tacaagtgaa ctagtaactt gtttattgca
      781 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
      841 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttagtc gatataactt
      901 cgtatagcat acattatacg aagttatgtc gacggtgtgg aaagtcccca ggctccccag
      961 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     1021 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     1081 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     1141 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     1201 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gaattcgcca
     1261 ccatgactga gtacaagcct actgtgcgtt tagccaccag ggatgatgtg ccacgagccg
     1321 tgaggacact agcagccgcc tttgctgact acccagctac aaggcacacc gtggaccctg
     1381 acagacacat tgaaagagtc acagaacttc aggagctgtt tttaactagg gtgggcctgg
     1441 atattggcaa agtctgggtg gccgatgatg gagcagcagt tgcagtgtgg accaccccag
     1501 agtctgttga agctggagct gtctttgcag aaattggacc aagaatggcc gagttatctg
     1561 gcagcaggct ggctgctcaa cagcagatgg aaggattatt agctccccac agacccaagg
     1621 agcctgcctg gttcctggca acagttggag tttctccaga ccatcagggc aagggcttag
     1681 gatctgctgt ggtgctgcct ggtgtggaag cagctgagag agctggggtg ccagcatttc
     1741 tggagacatc agctcctaga aacctgccct tctatgaacg gctgggcttc acagtgactg
     1801 ctgatgtgga ggtgcctgag ggccccagaa cctggtgcat gacaagaaaa ccaggagcct
     1861 gactgcagcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     1921 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     1981 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2041 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctcg
     2101 aggagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     2161 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     2221 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     2281 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     2341 gacgaaacac cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc
     2401 tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt
     2461 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2521 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2581 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2641 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2701 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2761 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2821 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2881 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2941 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3001 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3061 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3121 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3181 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3241 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3301 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3361 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3421 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3481 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3541 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3601 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3661 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3721 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3781 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3841 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3901 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3961 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4021 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4081 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4141 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4201 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4261 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4321 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4381 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4441 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     4501 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     4561 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     4621 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     4681 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa
     4741 tacataacct tatgtatcat acacatacga tttaggtgac actata
//
