LOCUS       sequence-228064-        4756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mKeima8.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4756)
  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 4756)
  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-P18
FEATURES             Location/Qualifiers
     source          1..4756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(64..66,67..726)
                     /codon_start=1
                     /product="human codon-optimized monomeric red fluorescent 
                     protein with a large Stokes shift (Guan et al. 2015)"
                     /label=hmKeima8.5
                     /translation="VSVIAEHMTYKVYMSGTVNGHYFEVEGDGEGKPYEGEQTVKLTVT
                     KGGPLPFAWHILSPQLQYGSIPFTKYPADIPDYFKQSFPEGYTWVRSMNFEDGAVCTVS
                     NDSSIRGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERMFARDGMLIGNDHMALKL
                     EGGGHYLCEFKSAYKARKPVRMPGLYEIDRKLDVTSHNRDYTSVEQREIAIARHPLLG"
     polyA_signal    736..857
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(773..792)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     827..846
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(864..897)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        904..1220
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1071..1206
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1842..2066
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2073..2321
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2073..2093
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2244..2263
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2323..2356)
                     /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(2393..2412)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2394..2412)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2499..2516)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2670..3258)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2750..2769)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3429..4220,4221..4289))
                     /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     4052..4071
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4290..4394)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4462..4480
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4518..4540)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4640..4659
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4740..4756,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4740..4756,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagcg tgatcgccga gcacatgacc tacaaggtgt acatgagcgg caccgtgaac
      121 ggccactact tcgaggtgga gggcgacggc gagggcaagc cctacgaggg cgagcagacc
      181 gtgaagctga ccgtgaccaa gggtggcccc ctgcccttcg cctggcacat cctgagcccc
      241 cagctccagt acggcagcat ccccttcacc aagtaccccg ccgacatccc cgactacttc
      301 aagcagagct tccccgaggg ctacacctgg gtgcgcagca tgaacttcga ggacggcgcc
      361 gtgtgcaccg tgagcaacga cagcagcatc cgcggcaact gcttcatcta caacgtgaag
      421 atcagcggcg agaacttccc ccccaacggc cccgtgatgc agaagaagac ccagggctgg
      481 gagcccagca ccgagcgcat gttcgcccgc gacggaatgc tgatcggcaa cgaccacatg
      541 gccctgaagc tggagggcgg cggccactac ctgtgcgagt tcaagagcgc ctacaaggcc
      601 cgcaagcccg tgaggatgcc cggcctgtac gagatcgacc gcaagctgga cgtgaccagc
      661 cacaaccgcg actacaccag cgtggagcag cgcgagatcg ccatcgcccg ccaccccctg
      721 ctgggctgaa ctagtaactt gtttattgca gcttataatg gttacaaata aagcaatagc
      781 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
      841 ctcatcaatg tatcttagtc gatataactt cgtatagcat acattatacg aagttatgtc
      901 gacggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
      961 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     1021 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     1081 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     1141 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     1201 gaggcctagg cttttgcaaa gaattcgcca ccatgactga gtacaagcct actgtgcgtt
     1261 tagccaccag ggatgatgtg ccacgagccg tgaggacact agcagccgcc tttgctgact
     1321 acccagctac aaggcacacc gtggaccctg acagacacat tgaaagagtc acagaacttc
     1381 aggagctgtt tttaactagg gtgggcctgg atattggcaa agtctgggtg gccgatgatg
     1441 gagcagcagt tgcagtgtgg accaccccag agtctgttga agctggagct gtctttgcag
     1501 aaattggacc aagaatggcc gagttatctg gcagcaggct ggctgctcaa cagcagatgg
     1561 aaggattatt agctccccac agacccaagg agcctgcctg gttcctggca acagttggag
     1621 tttctccaga ccatcagggc aagggcttag gatctgctgt ggtgctgcct ggtgtggaag
     1681 cagctgagag agctggggtg ccagcatttc tggagacatc agctcctaga aacctgccct
     1741 tctatgaacg gctgggcttc acagtgactg ctgatgtgga ggtgcctgag ggccccagaa
     1801 cctggtgcat gacaagaaaa ccaggagcct gactgcagcg actgtgcctt ctagttgcca
     1861 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     1921 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     1981 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     2041 tgctggggat gcggtgggct ctatggctcg aggagggcct atttcccatg attccttcat
     2101 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     2161 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2221 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2281 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgataacttc gtatagcata
     2341 cattatacga agttatggta ccgatgcagc tagcccgcgg atctgccggt ctccctatag
     2401 tgagtcgtat taatttcgat aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg
     2461 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2521 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2581 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2641 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2701 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2761 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2821 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     2881 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2941 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3001 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3061 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     3121 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3181 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3241 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3301 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3361 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3421 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3481 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3541 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3601 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3661 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3721 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3781 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3841 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3901 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3961 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4021 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4081 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4141 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4201 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4261 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4321 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4381 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     4441 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     4501 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4561 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4621 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac
     4681 atattgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga
     4741 tttaggtgac actata
//
