LOCUS       sequence-204754-        3614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3XFlag::TEV::6XHis::mTurquoise::6XHis::TEV::3XFlag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3614)
  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 3614)
  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: pAP639-2F
FEATURES             Location/Qualifiers
     source          1..3614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     243..265
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     257..274
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     258..274
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             301..366
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             367..387
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             388..405
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(406..408,409..1119)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2010)"
                     /label=mTurquoise
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISDNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(709..728)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1056..1077
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1120..1137
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1138..1158
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1159..1224
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1272..1288)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1272..1288)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1285..1307)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1296..1312
                     /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(1320..1326,1327..1344,1345..1350))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1365..1386
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1503..1520)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1674..2262)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1754..1773)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2433..3224,3225..3293))
                     /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     3056..3075
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3294..3398)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3466..3484
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3522..3544)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
       61 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
      121 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
      181 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
      241 ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta cccggggatc
      301 gattataaag atcatgatgg ggattataaa gatcatgata tagattataa agatgatgat
      361 gataaagaaa atttatattt tcagggtcat caccatcacc atcacgtgag caagggcgag
      421 gagctgttca ccggagtggt gcccatcctg gtcgagctgg acggagacgt aaacggccac
      481 aagttcagcg tgtccggaga gggcgagggc gatgccacct acggcaagct gaccctcaag
      541 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgtcc
      601 tggggcgtgc aatgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
      661 tccgccatgc ccgaaggata cgtccaggag cgcaccatct tcttcaagga cgacggcaac
      721 tacaagaccc gcgccgaggt gaagttcgag ggagacaccc tggtgaaccg catcgagctg
      781 aagggaatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
      841 atcagcgaca acgtctacat caccgccgac aagcagaaga acggcatcaa ggccaacttc
      901 aagatccgcc acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac
      961 acccccatcg gagacggccc cgtgctgctc cccgacaacc actacctgag cacccagtcc
     1021 aagctgtcca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1081 gccgccggga tcactctcgg catggacgag ctgtacaagc accatcacca tcaccatgag
     1141 aacctctact tccaaggaga ctacaaagac catgacggtg attataaaga tcatgatatc
     1201 gattacaagg atgacgatga caaggatcct ctagagtcga cctgcaggca tgcaagcttg
     1261 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     1321 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     1381 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1441 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1501 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1561 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1621 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1681 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1741 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1801 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1861 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1921 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1981 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2041 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2101 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2161 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2221 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2281 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2341 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2401 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2461 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2521 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2581 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     2641 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     2701 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     2761 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     2821 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     2881 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     2941 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3001 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3061 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3121 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3181 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3241 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3301 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3361 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3421 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     3481 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     3541 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     3601 gcgtcagcgg gtgt
//
