LOCUS       sequence-228140-        4411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with SNAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4411)
  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 4411)
  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-Z22
FEATURES             Location/Qualifiers
     source          1..4411
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..606
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for mammalian expression"
                     /translation="DKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVEV
                     PAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKV
                     VKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLAV
                     KEWLLAHEGHRLGKPGLG"
     polyA_signal    616..737
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(653..672)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     707..726
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        784..1100
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      951..1086
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1107..1116
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1113..1487
                     /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    1497..1721
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1728..1976
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1728..1748
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1899..1918
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1978..2011)
                     /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(2048..2067)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2049..2067)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2154..2171)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2325..2913)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2405..2424)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3084..3875,3876..3944))
                     /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     3707..3726
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3945..4049)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4117..4135
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4173..4195)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4295..4314
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4395..4411,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4395..4411,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgacaaag actgcgaaat gaagcgcacc accctggata gccctctggg caagctggaa
      121 ctgtctgggt gcgaacaggg cctgcacgag atcaagctgc tgggcaaagg aacatctgcc
      181 gccgacgccg tggaagtgcc tgccccagcc gccgtgctgg gcggaccaga gccactgatg
      241 caggccaccg cctggctcaa cgcctacttt caccagcctg aggccatcga ggagttccct
      301 gtgccagccc tgcaccaccc agtgttccag caggagagct ttacccgcca ggtgctgtgg
      361 aaactgctga aagtggtgaa gttcggagag gtcatcagct accagcagct ggccgccctg
      421 gccggcaatc ccgccgccac cgccgccgtg aaaaccgccc tgagcggaaa tcccgtgccc
      481 attctgatcc cctgccaccg ggtggtgtct agctctggcg ccgtgggggg ctacgagggc
      541 gggctcgccg tgaaagagtg gctgctggcc cacgagggcc acagactggg caagcctggg
      601 ctgggttgaa ctagtaactt gtttattgca gcttataatg gttacaaata aagcaatagc
      661 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
      721 ctcatcaatg tatcttagtc gatataactt cgtatagcat acattatacg aagttatgtc
      781 gacggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
      841 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
      901 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
      961 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     1021 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     1081 gaggcctagg cttttgcaaa gaattcgcca ccatggccaa gttgaccagt gccgttccgg
     1141 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
     1201 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca
     1261 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg
     1321 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct
     1381 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg
     1441 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgactg cagcgactgt
     1501 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1561 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1621 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1681 agacaatagc aggcatgctg gggatgcggt gggctctatg gctcgaggag ggcctatttc
     1741 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     1801 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     1861 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     1921 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgata
     1981 acttcgtata gcatacatta tacgaagtta tggtaccgat gcagctagcc cgcggatctg
     2041 ccggtctccc tatagtgagt cgtattaatt tcgataagcc aggttaacct gcattaatga
     2101 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2161 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2221 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2281 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2341 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2401 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2461 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     2521 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2581 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2641 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2701 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2761 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2821 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2881 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2941 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3001 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3061 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3121 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3181 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3241 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3301 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3361 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3421 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3481 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3541 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3601 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3661 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3721 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3781 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3841 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3901 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3961 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4021 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4081 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     4141 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4201 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     4261 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     4321 gtgcaccata tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt
     4381 atcatacaca tacgatttag gtgacactat a
//
