LOCUS       sequence-438494-        4726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint gene tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4726)
  AUTHORS   Fisch D, Zhang T, Sun H, Ma W, Tan Y, Gygi SP, Higgins DE, Kagan JC
  TITLE     Molecular definition of the endogenous Toll-like receptor signalling
            pathways.
  JOURNAL   Nature. 2024 Jul;631(8021):635-644. doi: 10.1038/s41586-024-07614-7.
            Epub 2024 Jul 3.
  PUBMED    38961291
REFERENCE   2  (bases 1 to 4726)
  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"; doi: 
            "10.1038/s41586-024-07614-7"; journalName: "Nature"; date: 
            "2024-07"; volume: "631"; issue: "8021"; pages: "635-644"
COMMENT     Sequence Label: pCRISPaint-APEX2-GFP-3xFLAG-PuroR
FEATURES             Location/Qualifiers
     source          1..4726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..49
                     /note="synthetic polyadenylation signal"
     misc_feature    63..154
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     103..122
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     CDS             218..964
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             join(965..967,968..970,971..1681)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGKELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1010..1031)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1271..1290)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1618..1639
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1682..1747
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1757..1810
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     polyA_signal    2434..2555
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2445..2464)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2499..2518
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2803..2820)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2974..3562)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3054..3073)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3762..4553,4554..4622))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta
       61 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc
      121 cccagtgcaa gtgcaggtgc cagaacattt ctcttaatga tagctagcgg gccagtaccc
      181 aaaaagcggg gggtctggtg gcagtggagg gggatccgga aagtcttacc caactgtgag
      241 tgctgattac caggacgccg ttgagaaggc gaagaagaag ctcagaggct tcatcgctga
      301 gaagagatgc gctcctctaa tgctccgttt ggcattccac tctgctggaa cctttgacaa
      361 gggcacgaag accggtggac ccttcggaac catcaagcac cctgccgaac tggctcacag
      421 cgctaacaac ggtcttgaca tcgctgttag gcttttggag ccactcaagg cggagttccc
      481 tattttgagc tacgccgatt tctaccagtt ggctggcgtt gttgccgttg aggtcacggg
      541 tggacctaag gttccattcc accctggaag agaggacaag cctgagccac caccagaggg
      601 tcgcttgccc gatcccacta agggttctga ccatttgaga gatgtgtttg gcaaagctat
      661 ggggcttact gaccaagata tcgttgctct atctgggggt cacactattg gagctgcaca
      721 caaggagcgt tctggatttg agggtccctg gacctctaat cctcttattt tcgacaactc
      781 atacttcacg gagttgttga gtggtgagaa ggaaggtctc cttcagctac cttctgacaa
      841 ggctcttttg tctgaccctg tattccgccc tctcgttgat aaatatgcag cggacgaaga
      901 tgccttcttt gctgattacg ctgaggctca ccaaaagctt tccgagcttg ggtttgctga
      961 tgccatggtg agcaagggca aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1021 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1081 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1141 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1201 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1261 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1321 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1381 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1441 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1501 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1561 ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat
     1621 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1681 ggattacaag gaccacgacg gcgattataa ggaccatgat attgattata aggacgatga
     1741 cgataagtcc ggaggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa
     1801 tcctggccca atgactgaat acaagcctac tgtcaggttg gctacaagag acgacgttcc
     1861 tagagccgtg agaactctgg ctgcagcctt cgccgactac cccgccacga gacacaccgt
     1921 tgacccagat cggcatattg agagagtgac tgaactgcag gagctgtttc ttacaagagt
     1981 tggcctcgac ataggcaagg tgtgggtggc ggacgacggc gccgccgtgg ccgtctggac
     2041 cactcccgaa tcagttgagg ctggcgccgt attcgctgag atcggcccga gaatggctga
     2101 gctcagcggg agtaggctcg cggcacagca gcaaatggag ggactgctgg caccacacag
     2161 gcccaaagaa cccgcctggt tcctggcaac cgtcggtgta tctcccgatc atcaggggaa
     2221 aggtctgggc tctgccgtag tgctccctgg cgtggaggca gctgagagag caggagtacc
     2281 tgccttcttg gagacctccg ctccaaggaa tcttcccttc tatgaacggt tgggcttcac
     2341 cgtgacagcc gacgtggaag tccccgaagg cccccgcact tggtgcatga cgaggaagcc
     2401 tggagcgtga gggcccgctt cgagcagaca tgataagata cattgatgag tttggacaaa
     2461 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     2521 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     2581 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     2641 gtggtaaaat cgataaagat ccgtcgaccg atgcccttga gagccttcaa cccagtcagc
     2701 tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc
     2761 atgcaactcg taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct
     2821 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2881 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2941 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3001 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3061 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3121 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3181 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3241 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3301 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3361 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3421 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3481 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3541 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3601 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3661 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3721 ttggtctgac agcggccgca aatgctaaac cactgcagtg gttaccagtg cttgatcagt
     3781 gaggcaccga tctcagcgat ctgcctattt cgttcgtcca tagtggcctg actccccgtc
     3841 gtgtagatca ctacgattcg tgagggctta ccatcaggcc ccagcgcagc aatgatgccg
     3901 cgagagccgc gttcaccggc ccccgatttg tcagcaatga accagccagc agggagggcc
     3961 gagcgaagaa gtggtcctgc tactttgtcc gcctccatcc agtctatgag ctgctgtcgt
     4021 gatgctagag taagaagttc gccagtgagt agtttccgaa gagttgtggc cattgctact
     4081 ggcatcgtgg tatcacgctc gtcgttcggt atggcttcgt tcaactctgg ttcccagcgg
     4141 tcaagccggg tcacatgatc acccatatta tgaagaaatg cagtcagctc cttagggcct
     4201 ccgatcgttg tcagaagtaa gttggccgcg gtgttgtcgc tcatggtaat ggcagcacta
     4261 cacaattctc ttaccgtcat gccatccgta agatgctttt ccgtgaccgg cgagtactca
     4321 accaagtcgt tttgtgagta gtgtatacgg cgaccaagct gctcttgccc ggcgtctata
     4381 cgggacaaca ccgcgccaca tagcagtact ttgaaagtgc tcatcatcgg gaatcgttct
     4441 tcggggcgga aagactcaag gatcttgccg ctattgagat ccagttcgat atagcccact
     4501 cttgcaccca gttgatcttc agcatctttt actttcacca gcgtttcggg gtgtgcaaaa
     4561 acaggcaagc aaaatgccgc aaagaaggga atgagtgcga cacgaaaatg ttggatgctc
     4621 atactcgtcc tttttcaata ttattgaagc atttatcagg gttactagta cgtctctcaa
     4681 ggataagtaa gtaatattaa ggtacgggag gtattggaca ggccgc
//
