LOCUS       sequence-204752-        3572 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEV::eGFP::ER::6xHis::linker::3XFlag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3572)
  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 3572)
  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: pAP685-2
FEATURES             Location/Qualifiers
     source          1..3572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..27
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(65..87)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     187..206
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     400..422
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     414..431
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     415..431
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             458..478
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(479..481,482..1192)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(509..537)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1132..1152
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1205..1222
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1274..1339
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1387..1403)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1387..1403)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1400..1422)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1411..1427
                     /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(1435..1441,1442..1459,1460..1465))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1480..1501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1618..1635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1789..2377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1869..1888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2548..3339,3340..3408))
                     /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     3171..3190
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3409..3513)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
       61 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
      121 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
      181 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
      241 acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact
      301 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
      361 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
      421 cgacggccag tgaattcgag ctcggtaccc ggggatcgag aacctctact tccaaggagt
      481 gagtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga
      541 tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa
      601 acttaccctt aaatttattt gcactactgg aaaactacct gttccatggc caacacttgt
      661 cactactctg acctacggcg tgcagtgctt cagccggtac cccgaccaca tgaagcagca
      721 tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa
      781 agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa
      841 tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt
      901 ggaatacaac tataactcac acaatgtata catcatggca gacaaacaaa agaatggaat
      961 caaagttaac ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca
     1021 ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct
     1081 gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca tggtccttct
     1141 tgagtttgta acagctgctg ggattacact cggcatggac gagctgtaca agtacgtagc
     1201 cggccatcat caccatcacc attgcccggg ggatcggtgg agctccaccg gtggcggccg
     1261 ctctagaact agtgactaca aagaccatga cggtgattat aaagatcatg atatcgatta
     1321 caaggatgac gatgacaagg atcctctaga gtcgacctgc aggcatgcaa gcttggcgta
     1381 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     1441 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     1501 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     1561 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     1621 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1681 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1741 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1801 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1861 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1921 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1981 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2041 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2101 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2161 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2221 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2281 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2341 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2401 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2461 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2521 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2581 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2641 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2701 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2761 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2821 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     2881 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2941 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3001 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3061 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     3121 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     3181 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3241 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3301 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3361 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     3421 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3481 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     3541 cgtctaagaa accattatta tcatgacatt aa
//
