LOCUS       sequence-228072-        5191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with YFP-LID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5191)
  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 5191)
  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-P25
FEATURES             Location/Qualifiers
     source          1..5191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(64..66,67..777)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(106..127)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(367..386)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     714..735
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             784..1104
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     polyA_signal    1171..1292
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1208..1227)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1262..1281
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1299..1332)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1339..1655
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1506..1641
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2277..2501
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2508..2756
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2508..2528
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2679..2698
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2758..2791)
                     /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(2828..2847)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2829..2847)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2934..2951)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3105..3693)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3185..3204)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3864..4655,4656..4724))
                     /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     4487..4506
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4725..4829)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4897..4915
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4953..4975)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5075..5094
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5175..5191,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5175..5191,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      121 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      181 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      241 ctcgtgacca ccttcggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag
      301 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      361 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      421 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      481 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      541 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      601 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      661 tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      721 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggaa
      781 ttcggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc
      841 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaagt tgattcctcc
      901 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg
      961 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat
     1021 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc
     1081 gatgtggagc ttctaaaact ggaaacgcgt ggagtggagg aagtggcgga gggggtagtt
     1141 ctcttaaggc ggcgaggtaa ttagactagt aacttgttta ttgcagctta taatggttac
     1201 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     1261 tgtggtttgt ccaaactcat caatgtatct tagtcgatat aacttcgtat agcatacatt
     1321 atacgaagtt atgtcgacgg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     1381 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     1441 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     1501 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     1561 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     1621 gaggaggctt ttttggaggc ctaggctttt gcaaagaatt cgccaccatg actgagtaca
     1681 agcctactgt gcgtttagcc accagggatg atgtgccacg agccgtgagg acactagcag
     1741 ccgcctttgc tgactaccca gctacaaggc acaccgtgga ccctgacaga cacattgaaa
     1801 gagtcacaga acttcaggag ctgtttttaa ctagggtggg cctggatatt ggcaaagtct
     1861 gggtggccga tgatggagca gcagttgcag tgtggaccac cccagagtct gttgaagctg
     1921 gagctgtctt tgcagaaatt ggaccaagaa tggccgagtt atctggcagc aggctggctg
     1981 ctcaacagca gatggaagga ttattagctc cccacagacc caaggagcct gcctggttcc
     2041 tggcaacagt tggagtttct ccagaccatc agggcaaggg cttaggatct gctgtggtgc
     2101 tgcctggtgt ggaagcagct gagagagctg gggtgccagc atttctggag acatcagctc
     2161 ctagaaacct gcccttctat gaacggctgg gcttcacagt gactgctgat gtggaggtgc
     2221 ctgagggccc cagaacctgg tgcatgacaa gaaaaccagg agcctgactg cagcgactgt
     2281 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2341 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2401 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2461 agacaatagc aggcatgctg gggatgcggt gggctctatg gctcgaggag ggcctatttc
     2521 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     2581 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     2641 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     2701 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgata
     2761 acttcgtata gcatacatta tacgaagtta tggtaccgat gcagctagcc cgcggatctg
     2821 ccggtctccc tatagtgagt cgtattaatt tcgataagcc aggttaacct gcattaatga
     2881 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2941 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3001 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3061 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3121 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3181 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3241 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     3301 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3361 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3421 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3481 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3541 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3601 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3661 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3721 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3781 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3841 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3901 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3961 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4021 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4081 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4141 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4201 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4261 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4321 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4381 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4441 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4501 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4561 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4621 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4681 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4741 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4801 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4861 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     4921 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4981 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     5041 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     5101 gtgcaccata tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt
     5161 atcatacaca tacgatttag gtgacactat a
//
