LOCUS       sequence-232617-        4774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mClover3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4774)
  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 4774)
  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-P08
FEATURES             Location/Qualifiers
     source          1..4774
                     /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"
     primer_bind     complement(123..144)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     731..752
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    771..892
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(808..827)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     862..881
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(899..932)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        939..1255
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1106..1241
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1877..2101
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2108..2356
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2108..2128
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2279..2298
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2358..2391)
                     /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(2428..2447)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2429..2447)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2534..2551)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2705..3293)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2785..2804)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3464..4255,4256..4324))
                     /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     4087..4106
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4325..4429)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4497..4515
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4553..4575)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4675..4694
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      121 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtccgc ggcgagggcg
      181 agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      241 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cgtggcctgc ttcagccgct
      301 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      361 aggagcgcac catctctttc aaggacgacg gtacctacaa gacccgcgcc gaggtgaagt
      421 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      481 gcaacatcct ggggcacaag ctggagtaca acttcaacag ccactacgtc tatatcacgg
      541 ccgacaagca gaagaactgc atcaaggcta acttcaagat ccgccacaac gttgaggacg
      601 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
      661 tgctgcccga caaccactac ctgagccatc agtccaagct gagcaaagac cccaacgaga
      721 agcgcgatca catggtcctg ctggagttcg tgaccgccgc ctgaactagt aacttgttta
      781 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
      841 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tagtcgatat
      901 aacttcgtat agcatacatt atacgaagtt atgtcgacgg tgtggaaagt ccccaggctc
      961 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     1021 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     1081 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     1141 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     1201 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagaatt
     1261 cgccaccatg actgagtaca agcctactgt gcgtttagcc accagggatg atgtgccacg
     1321 agccgtgagg acactagcag ccgcctttgc tgactaccca gctacaaggc acaccgtgga
     1381 ccctgacaga cacattgaaa gagtcacaga acttcaggag ctgtttttaa ctagggtggg
     1441 cctggatatt ggcaaagtct gggtggccga tgatggagca gcagttgcag tgtggaccac
     1501 cccagagtct gttgaagctg gagctgtctt tgcagaaatt ggaccaagaa tggccgagtt
     1561 atctggcagc aggctggctg ctcaacagca gatggaagga ttattagctc cccacagacc
     1621 caaggagcct gcctggttcc tggcaacagt tggagtttct ccagaccatc agggcaaggg
     1681 cttaggatct gctgtggtgc tgcctggtgt ggaagcagct gagagagctg gggtgccagc
     1741 atttctggag acatcagctc ctagaaacct gcccttctat gaacggctgg gcttcacagt
     1801 gactgctgat gtggaggtgc ctgagggccc cagaacctgg tgcatgacaa gaaaaccagg
     1861 agcctgactg cagcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     1921 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     1981 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2041 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     2101 gctcgaggag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     2161 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     2221 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     2281 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     2341 gaaaggacga aacaccgata acttcgtata gcatacatta tacgaagtta tggtaccgat
     2401 gcagctagcc cgcggatctg ccggtctccc tatagtgagt cgtattaatt tcgataagcc
     2461 aggttaacct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2521 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2581 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2641 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2701 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2761 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2821 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2881 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2941 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3001 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3061 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3121 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3181 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3241 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3301 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3361 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3421 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3481 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3541 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3601 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3661 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3721 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3781 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3841 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3901 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3961 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4021 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4081 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4141 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4201 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4261 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4321 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4381 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4441 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     4501 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     4561 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     4621 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     4681 cagagcagat tgtactgaga gtgcaccata tggacatatt gtcgttagaa cgcggctaca
     4741 attaatacat aaccttatgt atcatacaca tacg
//
