LOCUS       sequence-204753-        3524 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEV::linker::tagBFP::3XFlag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3524)
  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 3524)
  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: pAP975-1
FEATURES             Location/Qualifiers
     source          1..3524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..59
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     253..275
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     267..284
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     268..284
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             311..331
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             383..1078
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             1079..1144
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1192..1208)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1192..1208)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1205..1227)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1216..1232
                     /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(1240..1246,1247..1264,1265..1270))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1285..1306
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1423..1440)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1594..2182)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1674..1693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2353..3144,3145..3213))
                     /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     2976..2995
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3214..3318)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3386..3404
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3442..3464)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
       61 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
      121 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      181 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
      241 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta
      301 cccggggatc gagaacctct acttccaagg atgcccgggg gatcggtgga gctccaccgg
      361 tggcggccgc tctagaacta gtagcgagct gattaaggag aacatgcaca tgaagctgta
      421 catggagggc accgtggaca accatcactt caagtgcaca tccgaaggcg aaggcaagcc
      481 ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc
      541 cttcgacatc ctggcgacta gcttcctcta cggcagcaag accttcatca accacaccca
      601 gggcatcccc gacttcttca agcagtcctt ccctgaaggc ttcacatggg agagagtcac
      661 cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg
      721 cctcatatac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca
      781 gaagaaaaca ctcggctggg aggccttcac cgaaacgctg taccccgctg acggcggcct
      841 ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat
      901 caaaaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta
      961 tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca
     1021 cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatga
     1081 ctacaaagac catgacggtg attataaaga tcatgatatc gattacaagg atgacgatga
     1141 caaggatcct ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct
     1201 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     1261 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     1321 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1381 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     1441 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1501 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     1561 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1621 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1681 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1741 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1801 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1861 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1921 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1981 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2041 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2101 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2161 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2221 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2281 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2341 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2401 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2461 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     2521 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2581 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2641 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2701 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2761 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2821 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2881 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2941 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3001 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3061 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3121 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3181 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3241 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3301 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     3361 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     3421 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     3481 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgt
//
