LOCUS       sequence-232640-        4981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with Halo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4981)
  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 4981)
  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-P23
FEATURES             Location/Qualifiers
     source          1..4981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             81..968
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     polyA_signal    978..1099
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1015..1034)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1069..1088
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1106..1139)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1146..1462
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1313..1448
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2084..2308
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2315..2563
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2315..2335
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2486..2505
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2565..2598)
                     /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(2635..2654)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2636..2654)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2741..2758)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2912..3500)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2992..3011)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3671..4462,4463..4531))
                     /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     4294..4313
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4532..4636)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4704..4722
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4760..4782)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4882..4901
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gcagaaatcg gtactggctt tccattcgac ccccattatg
      121 tggaagtcct gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat ggcacccctg
      181 tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg
      241 ttgcaccgac ccatcgctgc attgctccag acctgatcgg tatgggcaaa tccgacaaac
      301 cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc atcgaagccc
      361 tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg ggtttccact
      421 gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc atccgcccta
      481 tcccgacctg ggacgaatgg ccagaatttg cccgcgagac cttccaggcc ttccgcacca
      541 ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt tatcgagggt acgctgccga
      601 tgggtgtcgt ccgcccgctg actgaagtcg agatggacca ttaccgcgag ccgttcctga
      661 atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc gccggtgagc
      721 cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag tcccctgtcc
      781 cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa gccgctcgcc
      841 tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg aatctgctgc
      901 aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctgtcc acgctcgaga
      961 tttccggctg aactagtaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     1021 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     1081 aactcatcaa tgtatcttag tcgatataac ttcgtatagc atacattata cgaagttatg
     1141 tcgacggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     1201 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     1261 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     1321 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     1381 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     1441 tggaggccta ggcttttgca aagaattcgc caccatgact gagtacaagc ctactgtgcg
     1501 tttagccacc agggatgatg tgccacgagc cgtgaggaca ctagcagccg cctttgctga
     1561 ctacccagct acaaggcaca ccgtggaccc tgacagacac attgaaagag tcacagaact
     1621 tcaggagctg tttttaacta gggtgggcct ggatattggc aaagtctggg tggccgatga
     1681 tggagcagca gttgcagtgt ggaccacccc agagtctgtt gaagctggag ctgtctttgc
     1741 agaaattgga ccaagaatgg ccgagttatc tggcagcagg ctggctgctc aacagcagat
     1801 ggaaggatta ttagctcccc acagacccaa ggagcctgcc tggttcctgg caacagttgg
     1861 agtttctcca gaccatcagg gcaagggctt aggatctgct gtggtgctgc ctggtgtgga
     1921 agcagctgag agagctgggg tgccagcatt tctggagaca tcagctccta gaaacctgcc
     1981 cttctatgaa cggctgggct tcacagtgac tgctgatgtg gaggtgcctg agggccccag
     2041 aacctggtgc atgacaagaa aaccaggagc ctgactgcag cgactgtgcc ttctagttgc
     2101 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2161 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2221 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2281 catgctgggg atgcggtggg ctctatggct cgaggagggc ctatttccca tgattccttc
     2341 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     2401 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     2461 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     2521 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgataact tcgtatagca
     2581 tacattatac gaagttatgg taccgatgca gctagcccgc ggatctgccg gtctccctat
     2641 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg
     2701 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2761 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2821 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2881 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2941 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3001 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3061 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3121 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3181 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3241 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3301 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3361 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3421 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3481 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3541 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3601 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3661 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3721 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3781 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3841 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3901 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3961 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4021 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4081 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4141 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4201 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4261 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4321 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4381 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4441 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4501 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4561 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4621 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     4681 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     4741 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     4801 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     4861 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg
     4921 acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac
     4981 g
//
