LOCUS       sequence-228151-        3913 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with S-Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3913)
  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 3913)
  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-Z32
FEATURES             Location/Qualifiers
     source          1..3913
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..108
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    118..239
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(155..174)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     209..228
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(246..279)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        286..602
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      453..588
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      609..618
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             615..989
                     /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    999..1223
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1230..1478
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1230..1250
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1401..1420
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1480..1513)
                     /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(1550..1569)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1551..1569)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1656..1673)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1827..2415)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1907..1926)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2586..3377,3378..3446))
                     /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     3209..3228
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3447..3551)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3619..3637
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3675..3697)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3797..3816
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3897..3913,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3897..3913,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagctg aactagtaac
      121 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
      181 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag
      241 tcgatataac ttcgtatagc atacattata cgaagttatg tcgacggtgt ggaaagtccc
      301 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
      361 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
      421 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
      481 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
      541 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
      601 aagaattcgc caccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg
      661 tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg
      721 acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc
      781 aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg
      841 ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg
      901 agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg
      961 tgcacttcgt ggccgaggag caggactgac tgcagcgact gtgccttcta gttgccagcc
     1021 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     1081 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     1141 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     1201 tggggatgcg gtgggctcta tggctcgagg agggcctatt tcccatgatt ccttcatatt
     1261 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     1321 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     1381 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     1441 tcttggcttt atatatcttg tggaaaggac gaaacaccga taacttcgta tagcatacat
     1501 tatacgaagt tatggtaccg atgcagctag cccgcggatc tgccggtctc cctatagtga
     1561 gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg
     1621 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     1681 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     1741 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1801 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1861 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1921 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1981 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat
     2041 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2101 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2161 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2221 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     2281 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2341 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2401 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2461 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     2521 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2581 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     2641 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     2701 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     2761 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     2821 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     2881 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     2941 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3001 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3061 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3121 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3181 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3241 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3301 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3361 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3421 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     3481 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3541 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     3601 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     3661 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     3721 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     3781 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatggacata
     3841 ttgtcgttag aacgcggcta caattaatac ataaccttat gtatcataca catacgattt
     3901 aggtgacact ata
//
