LOCUS       sequence-193080-        3596 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEV::eGFP::ER::Ollas::linker::3XFlag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3596)
  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 3596)
  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: pAP684-3
FEATURES             Location/Qualifiers
     source          1..3596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     134..153
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     387..404
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    521..542
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(557..562,563..580,581..587)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    595..611
                     /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     600..622
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     619..635
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     619..635
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(683..748)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(800..841)
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     CDS             complement(join(854..1564,1565..1567))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(894..914)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     1509..1537
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             complement(1568..1588)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1615..1632)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1615..1631)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1624..1646)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1840..1859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1959..1981
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2019..2037)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2105..2209
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2210..2278,2279..3070)
                     /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(2428..2447)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3241..3596,1..233)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
       61 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
      121 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
      181 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
      241 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
      301 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
      361 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
      421 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
      481 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      541 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      601 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cttgcatgcc
      661 tgcaggtcga ctctagagga tccttgtcat cgtcatcctt gtaatcgata tcatgatctt
      721 tataatcacc gtcatggtct ttgtagtcac tagttctaga gcggccgcca ccggtggagc
      781 tccaccgatc ccccgggcac tttcccatga gacgtggtcc gagctcgttg gcgaatccgg
      841 agccggctac gtacttgtac agctcgtcca tgccgagtgt aatcccagca gctgttacaa
      901 actcaagaag gaccatgtgg tctctctttt cgttgggatc tttcgaaagg gcagattgtg
      961 tggacaggta atggttgtct ggtaaaagga cagggccatc gccaattgga gtattttgtt
     1021 gataatggtc tgctagttga acgcttccat cttcaatgtt gtgtctaatt ttgaagttaa
     1081 ctttgattcc attcttttgt ttgtctgcca tgatgtatac attgtgtgag ttatagttgt
     1141 attccaattt gtgtccaaga atgtttccat cttctttaaa gtcaatacct tttaactcga
     1201 ttctattaac aagggtatca ccttcaaact tgacttcagc acgtgtcttg tagttcccgt
     1261 catctttgaa aaatatagtt ctttcctgta cataaccttc gggcatggca ctcttgaaaa
     1321 agtcatgctg cttcatgtgg tcggggtacc ggctgaagca ctgcacgccg taggtcagag
     1381 tagtgacaag tgttggccat ggaacaggta gttttccagt agtgcaaata aatttaaggg
     1441 taagttttcc gtatgttgca tcaccttcac cctctccact gacagaaaat ttgtgcccat
     1501 taacatcacc atctaattca acaagaattg ggacaactcc agtgaaaagt tcttctcctt
     1561 tactcactcc ttggaagtag aggttctcga tccccgggta ccgagctcga attcactggc
     1621 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     1681 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     1741 ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca
     1801 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     1861 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     1921 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     1981 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     2041 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     2101 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2161 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2221 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2281 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2341 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2401 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     2461 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2521 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2581 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2641 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2701 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     2761 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2821 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2881 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     2941 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3001 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3061 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3121 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3181 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3241 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3301 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3361 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     3421 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3481 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3541 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaa
//
