LOCUS       sequence-228136-        4531 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mKeima8.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4531)
  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 4531)
  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-Z18
FEATURES             Location/Qualifiers
     source          1..4531
                     /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"
     regulatory      1227..1236
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1233..1607
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1617..1841
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1848..2096
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1848..1868
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2019..2038
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2098..2131)
                     /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(2168..2187)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2169..2187)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2274..2291)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2445..3033)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2525..2544)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3204..3995,3996..4064))
                     /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     3827..3846
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4065..4169)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4237..4255
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4293..4315)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4415..4434
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4515..4531,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4515..4531,1)
                     /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 ccatggccaa gttgaccagt gccgttccgg
     1261 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
     1321 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca
     1381 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg
     1441 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct
     1501 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg
     1561 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgactg cagcgactgt
     1621 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1681 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1741 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1801 agacaatagc aggcatgctg gggatgcggt gggctctatg gctcgaggag ggcctatttc
     1861 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     1921 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     1981 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     2041 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgata
     2101 acttcgtata gcatacatta tacgaagtta tggtaccgat gcagctagcc cgcggatctg
     2161 ccggtctccc tatagtgagt cgtattaatt tcgataagcc aggttaacct gcattaatga
     2221 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2281 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2341 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2401 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2461 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2521 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2581 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     2641 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2701 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2761 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2821 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2881 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2941 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3001 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3061 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3121 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3181 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3241 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3301 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3361 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3421 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3481 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3541 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3601 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3661 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3721 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3781 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3841 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3901 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3961 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4021 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4081 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4141 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4201 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     4261 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4321 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     4381 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     4441 gtgcaccata tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt
     4501 atcatacaca tacgatttag gtgacactat a
//
