LOCUS       sequence-228080-        4135 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with V5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4135)
  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 4135)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-P31
FEATURES             Location/Qualifiers
     source          1..4135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..105
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    115..236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(152..171)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     206..225
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(243..276)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        283..599
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      450..585
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1221..1445
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1452..1700
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1452..1472
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1623..1642
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1702..1735)
                     /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(1772..1791)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1773..1791)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1878..1895)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2049..2637)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2129..2148)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2808..3599,3600..3668))
                     /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     3431..3450
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3669..3773)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3841..3859
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3897..3919)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4019..4038
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4119..4135,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4119..4135,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgtgaac tagtaacttg
      121 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
      181 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttagtcg
      241 atataacttc gtatagcata cattatacga agttatgtcg acggtgtgga aagtccccag
      301 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
      361 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
      421 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
      481 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
      541 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
      601 aattcgccac catgactgag tacaagccta ctgtgcgttt agccaccagg gatgatgtgc
      661 cacgagccgt gaggacacta gcagccgcct ttgctgacta cccagctaca aggcacaccg
      721 tggaccctga cagacacatt gaaagagtca cagaacttca ggagctgttt ttaactaggg
      781 tgggcctgga tattggcaaa gtctgggtgg ccgatgatgg agcagcagtt gcagtgtgga
      841 ccaccccaga gtctgttgaa gctggagctg tctttgcaga aattggacca agaatggccg
      901 agttatctgg cagcaggctg gctgctcaac agcagatgga aggattatta gctccccaca
      961 gacccaagga gcctgcctgg ttcctggcaa cagttggagt ttctccagac catcagggca
     1021 agggcttagg atctgctgtg gtgctgcctg gtgtggaagc agctgagaga gctggggtgc
     1081 cagcatttct ggagacatca gctcctagaa acctgccctt ctatgaacgg ctgggcttca
     1141 cagtgactgc tgatgtggag gtgcctgagg gccccagaac ctggtgcatg acaagaaaac
     1201 caggagcctg actgcagcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     1261 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     1321 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     1381 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     1441 tatggctcga ggagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     1501 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     1561 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     1621 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     1681 tgtggaaagg acgaaacacc gataacttcg tatagcatac attatacgaa gttatggtac
     1741 cgatgcagct agcccgcgga tctgccggtc tccctatagt gagtcgtatt aatttcgata
     1801 agccaggtta acctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1861 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     1921 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1981 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2041 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2101 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2161 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2221 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     2281 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2341 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2401 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2461 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     2521 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2581 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2641 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2701 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2761 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2821 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     2881 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     2941 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3001 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3061 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3121 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3181 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3241 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3301 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3361 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3421 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3481 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3541 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3601 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3661 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     3721 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     3781 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     3841 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     3901 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     3961 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     4021 gcatcagagc agattgtact gagagtgcac catatggaca tattgtcgtt agaacgcggc
     4081 tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca ctata
//
