LOCUS       sequence-193143-        3542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEV::linker::meGFP:3XFlag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3542)
  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 3542)
  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: pAP973-1
FEATURES             Location/Qualifiers
     source          1..3542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(154..742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(234..253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(913..1704,1705..1773))
                     /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     1536..1555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1774..1878)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1946..1964
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2002..2024)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2124..2143
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2337..2359
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2351..2368
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2352..2368
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2395..2415
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2497..2525)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3120..3140
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3181..3246
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3294..3310)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3294..3310)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3307..3329)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3318..3334
                     /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(3342..3348,3349..3366,3367..3372))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3387..3408
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3525..3542)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
       61 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
      121 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
      181 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
      241 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
      301 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
      361 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
      421 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
      481 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
      541 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
      601 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
      661 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
      721 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
      781 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
      841 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
      901 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
      961 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     1021 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     1081 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     1141 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     1201 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     1261 tatggcctca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     1321 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     1381 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     1441 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     1501 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     1561 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     1621 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     1681 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     1741 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     1801 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     1861 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     1921 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     1981 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     2041 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     2101 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     2161 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc
     2221 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     2281 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     2341 gtcacgacgt tgtaaaacga cggccagtga attcgagctc ggtacccggg gatcgagaac
     2401 ctctacttcc aaggatgccc gggggatcgg tggagctcca ccggtggcgg ccgctctaga
     2461 actagtgtga gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta
     2521 gatggtgatg ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca
     2581 tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca
     2641 acacttgtca ctactctgac ctacggcgtg cagtgcttca gccggtaccc cgaccacatg
     2701 aagcagcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata
     2761 tttttcaaag atgacgggaa ctacaagaca cgtgctgaag tcaagtttga aggtgatacc
     2821 cttgttaata gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga
     2881 cacaaattgg aatacaacta taactcacac aatgtataca tcatggcaga caaacaaaag
     2941 aatggaatca aagttaactt caaaattaga cacaacattg aagatggaag cgttcaacta
     3001 gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac
     3061 cattacctgt ccacacaatc taagctttcg aaagatccca acgaaaagag agaccacatg
     3121 gtccttcttg agtttgtaac agctgctggg attacactcg gcatggacga gctgtacaag
     3181 gactacaaag accatgacgg tgattataaa gatcatgata tcgattacaa ggatgacgat
     3241 gacaaggatc ctctagagtc gacctgcagg catgcaagct tggcgtaatc atggtcatag
     3301 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3361 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     3421 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     3481 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3541 ct
//
