LOCUS       sequence-228028-        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"
     CDS             64..951
                     /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    961..1082
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(998..1017)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1052..1071
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1089..1122)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1129..1377
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1129..1149
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1300..1319
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1379..1412)
                     /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(1449..1468)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1450..1468)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1555..1572)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1726..2314)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1806..1825)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2485..3276,3277..3345))
                     /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     3108..3127
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3346..3450)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3518..3536
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3574..3596)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3696..3715
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3796..3812,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3796..3812,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgcagaaa tcggtactgg ctttccattc gacccccatt atgtggaagt cctgggcgag
      121 cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt
      181 aacccgacct cctcctacgt gtggcgcaac atcatcccgc atgttgcacc gacccatcgc
      241 tgcattgctc cagacctgat cggtatgggc aaatccgaca aaccagacct gggttatttc
      301 ttcgacgacc acgtccgctt catggatgcc ttcatcgaag ccctgggtct ggaagaggtc
      361 gtcctggtca ttcacgactg gggctccgct ctgggtttcc actgggccaa gcgcaatcca
      421 gagcgcgtca aaggtattgc atttatggag ttcatccgcc ctatcccgac ctgggacgaa
      481 tggccagaat ttgcccgcga gaccttccag gccttccgca ccaccgacgt cggccgcaag
      541 ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg
      601 ctgactgaag tcgagatgga ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag
      661 ccactgtggc gcttcccaaa cgagctgcca atcgccggtg agccagcgaa catcgtcgcg
      721 ctggtcgaag aatacatgga ctggctgcac cagtcccctg tcccgaagct gctgttctgg
      781 ggcaccccag gcgttctgat cccaccggcc gaagccgctc gcctggccaa aagcctgcct
      841 aactgcaagg ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga caacccggac
      901 ctgatcggca gcgagatcgc gcgctggctg tcgacgctcg agatttccgg ctgaactagt
      961 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1021 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1081 tagtcgatat aacttcgtat agcatacatt atacgaagtt atgtcgagga gggcctattt
     1141 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
     1201 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     1261 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     1321 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgat
     1381 aacttcgtat agcatacatt atacgaagtt atggtaccga tgcagctagc ccgcggatct
     1441 gccggtctcc ctatagtgag tcgtattaat ttcgataagc caggttaacc tgcattaatg
     1501 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     1561 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     1621 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     1681 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     1741 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     1801 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     1861 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     1921 atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     1981 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2041 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2101 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     2161 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     2221 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     2281 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     2341 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     2401 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     2461 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     2521 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2581 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     2641 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     2701 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     2761 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     2821 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2881 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2941 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3001 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3061 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     3121 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     3181 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     3241 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3301 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3361 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     3421 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     3481 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     3541 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     3601 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     3661 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     3721 agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg
     3781 tatcatacac atacgattta ggtgacacta ta
//
