LOCUS       sequence-232600-        3812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with Halo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3812)
  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 3812)
  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-23
FEATURES             Location/Qualifiers
     source          1..3812
                     /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..1394
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1146..1166
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1317..1336
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1396..1429)
                     /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(1466..1485)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1467..1485)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1572..1589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1743..2331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1823..1842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2502..3293,3294..3362))
                     /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     3125..3144
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3363..3467)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3535..3553
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3591..3613)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3713..3732
                     /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 tcgaggaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     1201 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     1261 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     1321 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     1381 aaggacgaaa caccgataac ttcgtatagc atacattata cgaagttatg gtaccgatgc
     1441 agctagcccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag
     1501 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1561 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1621 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1681 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1741 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1801 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1861 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1921 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1981 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2041 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2101 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2161 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2221 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2281 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2341 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2401 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2461 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2521 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2581 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2641 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2701 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2761 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2821 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2881 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2941 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3001 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3061 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3121 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3181 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3241 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3301 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3361 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3421 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3481 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     3541 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     3601 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     3661 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     3721 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat
     3781 taatacataa ccttatgtat catacacata cg
//
