LOCUS       sequence-192946-        3653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3XFlag::TEV::V5::mOrange2::V5::TEV::3XFlag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3653)
  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 3653)
  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: pAP640-2
FEATURES             Location/Qualifiers
     source          1..3653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     385..402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    519..540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(555..560,561..578,579..585)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    593..609
                     /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)."
     primer_bind     598..620
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     617..633
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     617..633
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(681..746)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(747..767)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(768..809)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1515..1556)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1557..1577)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1578..1643)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1670..1687)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1670..1686)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1679..1701)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1895..1914)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2014..2036
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2074..2092)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2160..2264
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2265..2333,2334..3125)
                     /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     complement(2483..2502)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3296..3653,1..231)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
       61 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      301 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      361 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      421 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      481 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      541 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      601 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct tgcatgcctg
      661 caggtcgact ctagaggatc cttgtcatcg tcatccttgt aatcgatatc atgatcttta
      721 taatcaccgt catggtcttt gtagtctcct tggaagtaga ggttctcggt ggagtcgagt
      781 ccgaggagtg ggtttgggat tggctttccc ttgtagagct cgtccattcc tccggtggag
      841 tggcgtccct cggcacgttc gtattgctcc acgatggtgt agtcctcgtt gtgggaggtg
      901 atgtcgagct tgatgtcgac gatgtaggct cctgggagtt ggactggctt cttggccttg
      961 taggtggtct tgacctcgga ggtgtagtgt cctccgtcct tgagcttgag cctcatcttg
     1021 atctttccct tcagggctcc gtcctctggg tacatccgct cggaggaggc ctcccatccc
     1081 atggtcttct tttgcatgac tgggccgtcg gatgggaagt tggttccacg gagcttcacc
     1141 ttgtagatga actctccgtc ttggagggag gagtcttggg tgacggtgac cactcctccg
     1201 tcctcgtagt tcatgacacg ctcccacttg aatccctcgg ggaaggagag cttgaagtag
     1261 tctgggatgt cggctgggtg cttgacgtag gccttggagc cgtaggtgaa gtgtggggac
     1321 aggatgtccc aggcgaatgg gaggggtcct cccttggtga ccttgagctt ggcggtctgg
     1381 aatccctcgt atggacgtcc ctcgccctct ccctcgatct cgaactcgtg tccgttgacg
     1441 gatccctcca tacggacctt gaaacgcatg aactccttga tgatggccat gttattctcc
     1501 tctcccttgg agacagtact atctagaccg aggagagggt tagggatagg cttaccaccc
     1561 tgaaaatata aattttcttt atcatcatca tctttataat ctatatcatg atctttataa
     1621 tccccatcat gatctttata atcgatcccc gggtaccgag ctcgaattca ctggccgtcg
     1681 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     1741 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     1801 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     1861 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     1921 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     1981 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     2041 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     2101 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2161 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2221 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2281 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2341 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2401 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2461 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     2521 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2581 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2641 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2701 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2761 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     2821 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2881 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2941 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3001 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3061 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3121 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3181 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3241 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3301 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3361 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3421 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     3481 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3541 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3601 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acg
//
