LOCUS       sequence-228143-        4966 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with YFP-LID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4966)
  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 4966)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Z25
FEATURES             Location/Qualifiers
     source          1..4966
                     /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"
     regulatory      1662..1671
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1668..2042
                     /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    2052..2276
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2283..2531
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2283..2303
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2454..2473
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2533..2566)
                     /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(2603..2622)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2604..2622)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2709..2726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2880..3468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2960..2979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3639..4430,4431..4499))
                     /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     4262..4281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4500..4604)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4672..4690
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4728..4750)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4850..4869
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4950..4966,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4950..4966,1)
                     /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 gccaagttga
     1681 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
     1741 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
     1801 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
     1861 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
     1921 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
     1981 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
     2041 gactgcagcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2101 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2161 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2221 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctcg
     2281 aggagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     2341 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     2401 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     2461 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     2521 gacgaaacac cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc
     2581 tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt
     2641 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2701 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2761 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2821 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2881 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2941 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3001 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3061 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3121 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3181 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3241 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3301 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3361 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3421 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3481 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3541 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3601 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3661 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3721 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3781 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3841 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3901 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3961 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4021 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4081 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4141 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4201 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4261 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4321 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4381 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4441 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4501 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4561 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4621 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     4681 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     4741 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     4801 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     4861 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa
     4921 tacataacct tatgtatcat acacatacga tttaggtgac actata
//
