LOCUS       sequence-228070-        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"
     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"
     polyA_signal    2067..2291
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2298..2546
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2298..2318
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2469..2488
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2548..2581)
                     /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(2618..2637)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2619..2637)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2724..2741)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2895..3483)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2975..2994)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3654..4445,4446..4514))
                     /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     4277..4296
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4515..4619)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4687..4705
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4743..4765)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4865..4884
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4965..4981,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4965..4981,1)
                     /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 actgagtaca agcctactgt gcgtttagcc accagggatg
     1501 atgtgccacg agccgtgagg acactagcag ccgcctttgc tgactaccca gctacaaggc
     1561 acaccgtgga ccctgacaga cacattgaaa gagtcacaga acttcaggag ctgtttttaa
     1621 ctagggtggg cctggatatt ggcaaagtct gggtggccga tgatggagca gcagttgcag
     1681 tgtggaccac cccagagtct gttgaagctg gagctgtctt tgcagaaatt ggaccaagaa
     1741 tggccgagtt atctggcagc aggctggctg ctcaacagca gatggaagga ttattagctc
     1801 cccacagacc caaggagcct gcctggttcc tggcaacagt tggagtttct ccagaccatc
     1861 agggcaaggg cttaggatct gctgtggtgc tgcctggtgt ggaagcagct gagagagctg
     1921 gggtgccagc atttctggag acatcagctc ctagaaacct gcccttctat gaacggctgg
     1981 gcttcacagt gactgctgat gtggaggtgc ctgagggccc cagaacctgg tgcatgacaa
     2041 gaaaaccagg agcctgactg cagcgactgt gccttctagt tgccagccat ctgttgtttg
     2101 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2161 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     2221 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     2281 gggctctatg gctcgaggag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2341 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     2401 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2461 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2521 atatcttgtg gaaaggacga aacaccgata acttcgtata gcatacatta tacgaagtta
     2581 tggtaccgat gcagctagcc cgcggatctg ccggtctccc tatagtgagt cgtattaatt
     2641 tcgataagcc aggttaacct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2701 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2761 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2821 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2881 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2941 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3001 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3061 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     3121 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3181 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3241 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3301 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     3361 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3421 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3481 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3541 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3601 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3661 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3721 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3781 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3841 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3901 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3961 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4021 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4081 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4141 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4201 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4261 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4321 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4381 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4441 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4501 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4561 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4621 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     4681 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     4741 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     4801 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     4861 tatgcggcat cagagcagat tgtactgaga gtgcaccata tggacatatt gtcgttagaa
     4921 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
     4981 a
//
