LOCUS       sequence-204755-        3500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3XFlag::Topaz::Myc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3500)
  AUTHORS   Paix A, Folkmann A, Seydoux G
  TITLE     Precision genome editing using CRISPR-Cas9 and linear repair 
            templates in C. elegans.
  JOURNAL   Methods. 2017 May 15;121-122:86-93. doi: 
            10.1016/j.ymeth.2017.03.023. Epub 2017 Apr 7.
  PUBMED    28392263
REFERENCE   2  (bases 1 to 3500)
  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: "Methods."; 
            date: "2017-05-15"; volume: "121-122:86-93. doi"; pages: " 
            10.1016/j.ymeth.2017.03.023. Epub 2017 Apr 7"
COMMENT     Sequence Label: pAP574-1
FEATURES             Location/Qualifiers
     source          1..3500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..32
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     226..248
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     240..257
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     241..257
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             284..349
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(350..352,353..1063)
                     /codon_start=1
                     /product="enhanced yellow variant of GFP"
                     /label=Topaz YFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(392..413)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(653..672)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1000..1021
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1064..1093
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1141..1157)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1141..1157)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1154..1176)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1165..1181
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1189..1195,1196..1213,1214..1219))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1234..1255
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1372..1389)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1543..2131)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1623..1642)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2302..3093,3094..3162))
                     /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     2925..2944
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3163..3267)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3335..3353
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3391..3413)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
       61 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc
      121 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
      181 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
      241 gtaaaacgac ggccagtgaa ttcgagctcg gtacccgggg atcgactaca aagaccatga
      301 cggtgattat aaagatcatg atatcgatta caaggatgac gatgacaagg tgagcaaggg
      361 cgaggagctg ttcaccgggg tggtgcccat actggtcgag ctggacggcg acgtaaacgg
      421 ccacaagttc agtgtgtccg gcgagggcga gggcgatgcc acctacggca agctaaccct
      481 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccacgctcg tgaccacctt
      541 cggctacggc gtgcagtgct tcgcccgcta ccccgaccac atgcgccagc acgacttctt
      601 caagtccgcc atgcccgaag gctatgtcca ggagcgcacc atcttcttca aggacgacgg
      661 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctagtga accgcatcga
      721 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
      781 ctataacagc cacaacgtct atatcatggc cgacaagcag aagaacggca taaaggtgaa
      841 cttcaagatc cgccacaaca tcgaggacgg gagcgtgcag ctcgccgacc actaccagca
      901 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca
      961 gtccgcccta agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1021 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaggaacaaa aactgatatc
     1081 agaagaggat ctggatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg
     1141 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     1201 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     1261 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1321 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1381 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1441 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1501 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1561 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1621 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1681 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1741 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1801 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1861 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1921 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1981 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2041 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2101 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2161 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2221 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2281 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2341 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2401 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2461 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2521 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2581 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2641 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2701 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2761 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     2821 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     2881 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     2941 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3001 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3061 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3121 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     3181 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3241 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     3301 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     3361 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3421 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3481 tgttggcggg tgtcggggct
//
