LOCUS       sequence-193081-        3542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3XFlag::meGFP::linker::TEV.
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: pAP996-1
FEATURES             Location/Qualifiers
     source          1..3542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..42)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(196..784)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(276..295)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(955..1746,1747..1815))
                     /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     1578..1597
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1816..1920)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1988..2006
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2044..2066)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2166..2185
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2379..2401
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2393..2410
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2394..2410
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2437..2502
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2533..2561)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3156..3176
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3268..3288
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3336..3352)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3336..3352)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3349..3371)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3360..3376
                     /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(3384..3390,3391..3408,3409..3414))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3429..3450
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
       61 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      121 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      181 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      241 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      301 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      361 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      421 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      481 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      541 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      601 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
      661 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      721 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      781 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      841 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
      901 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
      961 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1021 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1081 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1141 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1201 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1261 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1321 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1381 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1441 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1501 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1561 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1621 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     1681 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     1741 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     1801 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     1861 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     1921 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     1981 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     2041 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     2101 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     2161 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     2221 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     2281 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     2341 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     2401 gacggccagt gaattcgagc tcggtacccg gggatcgact acaaagacca tgacggtgat
     2461 tataaagatc atgatatcga ttacaaggat gacgatgaca aggtgagtaa aggagaagaa
     2521 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa
     2581 ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt
     2641 atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac tctgacctac
     2701 ggcgtgcagt gcttcagccg gtaccccgac cacatgaagc agcatgactt tttcaagagt
     2761 gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac
     2821 aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa
     2881 ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac
     2941 tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa
     3001 attagacaca acattgaaga tggaagcgtt caactagcag accattatca acaaaatact
     3061 ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac acaatctaag
     3121 ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct
     3181 gctgggatta cactcggcat ggacgagctg tacaagtgcc cgggggatcg gtggagctcc
     3241 accggtggcg gccgctctag aactagtgag aacctctact tccaaggaga tcctctagag
     3301 tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3361 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3421 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3481 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3541 tt
//
