LOCUS       sequence-228141-        4756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with Halo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4756)
  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 4756)
  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-Z23
FEATURES             Location/Qualifiers
     source          1..4756
                     /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..1445
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1296..1431
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1452..1461
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1458..1832
                     /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    1842..2066
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2073..2321
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2073..2093
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2244..2263
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2323..2356)
                     /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(2393..2412)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2394..2412)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2499..2516)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2670..3258)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2750..2769)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3429..4220,4221..4289))
                     /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     4052..4071
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4290..4394)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4462..4480
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4518..4540)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4640..4659
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4740..4756,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4740..4756,1)
                     /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 atgtcgacgg tgtggaaagt
     1141 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     1201 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     1261 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     1321 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     1381 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     1441 gcaaagaatt cgccaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg
     1501 acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg
     1561 aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg
     1621 accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt
     1681 acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga
     1741 ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact
     1801 gcgtgcactt cgtggccgag gagcaggact gactgcagcg actgtgcctt ctagttgcca
     1861 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     1921 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     1981 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     2041 tgctggggat gcggtgggct ctatggctcg aggagggcct atttcccatg attccttcat
     2101 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     2161 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2221 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2281 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgataacttc gtatagcata
     2341 cattatacga agttatggta ccgatgcagc tagcccgcgg atctgccggt ctccctatag
     2401 tgagtcgtat taatttcgat aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg
     2461 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2521 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2581 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2641 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2701 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2761 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2821 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     2881 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2941 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3001 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3061 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     3121 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3181 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3241 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3301 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3361 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3421 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3481 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3541 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3601 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3661 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3721 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3781 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3841 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3901 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3961 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4021 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4081 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4141 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4201 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4261 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4321 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4381 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     4441 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     4501 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4561 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4621 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac
     4681 atattgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga
     4741 tttaggtgac actata
//
