LOCUS       sequence-193118-        3581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEV::eGFP::ER::HA::linker::3XFlag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3581)
  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 3581)
  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: pAP683-2
FEATURES             Location/Qualifiers
     source          1..3581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..46)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(200..788)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(280..299)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(959..1750,1751..1819))
                     /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     1582..1601
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1820..1924)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1992..2010
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2048..2070)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2170..2189
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2383..2405
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2397..2414
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2398..2414
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2441..2461
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2462..2464,2465..3175)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2492..2520)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3115..3135
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3188..3214
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3266..3331
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3379..3395)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3379..3395)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3392..3414)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3403..3419
                     /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(3427..3433,3434..3451,3452..3457))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3472..3493
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
       61 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
      121 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
      181 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
      241 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      301 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      361 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      421 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      481 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      541 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      601 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
      661 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
      721 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
      781 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
      841 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
      901 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
      961 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     1021 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     1081 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     1141 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     1201 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     1261 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     1321 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     1381 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     1441 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     1501 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     1561 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     1621 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     1681 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     1741 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     1801 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     1861 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     1921 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     1981 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     2041 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     2101 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     2161 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
     2221 cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca
     2281 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     2341 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     2401 aaacgacggc cagtgaattc gagctcggta cccggggatc gagaacctct acttccaagg
     2461 agtgagtaaa ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg
     2521 tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg
     2581 aaaacttacc cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact
     2641 tgtcactact ctgacctacg gcgtgcagtg cttcagccgg taccccgacc acatgaagca
     2701 gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt
     2761 caaagatgac gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt
     2821 taatagaatc gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa
     2881 attggaatac aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg
     2941 aatcaaagtt aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga
     3001 ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta
     3061 cctgtccaca caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct
     3121 tcttgagttt gtaacagctg ctgggattac actcggcatg gacgagctgt acaagtacgt
     3181 agccggctac ccatatgatg tcccggatta cgcttgcccg ggggatcggt ggagctccac
     3241 cggtggcggc cgctctagaa ctagtgacta caaagaccat gacggtgatt ataaagatca
     3301 tgatatcgat tacaaggatg acgatgacaa ggatcctcta gagtcgacct gcaggcatgc
     3361 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3421 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     3481 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3541 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc g
//
