LOCUS       sequence-193114-        3536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3XFlag::tagRFP::linker::TEV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3536)
  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 3536)
  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: pAP997-1
FEATURES             Location/Qualifiers
     source          1..3536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(162..750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(242..261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(921..1712,1713..1781))
                     /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     1544..1563
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1782..1886)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1954..1972
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2010..2032)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2132..2151
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2345..2367
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2359..2376
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2360..2376
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2403..2468
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2469..3176
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             3228..3248
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3296..3312)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3296..3312)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3309..3331)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3320..3336
                     /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(3344..3350,3351..3368,3369..3374))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3389..3410
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(join(3527..3536,1..8))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
       61 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      121 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      181 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      241 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      301 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      361 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      421 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      481 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      541 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      601 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
      661 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
      721 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
      781 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
      841 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
      901 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
      961 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1021 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     1081 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     1141 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     1201 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     1261 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     1321 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     1381 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     1441 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     1501 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     1561 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     1621 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     1681 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     1741 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     1801 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     1861 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     1921 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccccttcgt
     1981 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     2041 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     2101 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     2161 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     2221 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     2281 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     2341 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tcgagctcgg tacccgggga
     2401 tcgactacaa agaccatgac ggtgattata aagatcatga tatcgattac aaggatgacg
     2461 atgacaaggt gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca
     2521 tggagggcac cgtgaacaac caccacttca aatgcacatc cgagggcgaa ggcaagccct
     2581 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
     2641 ttgacatcct ggctaccagc ttcatgtacg gcagcagaac cttcatcaac cacacccagg
     2701 gcatccccga cttctttaag cagtcctttc ctgagggctt cacatgggag agagtcacca
     2761 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgtc
     2821 tcatctacaa cgtcaagatc agaggggtga acttcccatc caacggccct gtgatgcaga
     2881 agaaaacact cggctgggag gccaataccg agatgctgta ccccgctgac ggcggcctgg
     2941 aaggcagaag cgacatggcc ctgaagctcg tgggcggggg ccacctgatc tgcaatttca
     3001 agaccacata cagatccaag aaacccgcta agaacctcaa gatgcccggc gtctactatg
     3061 tggaccacag actggaaaga atcaaagagg ccgacaaaga gacctacgtc gagcagcacg
     3121 aggtggctgt ggccagatac tgcgacctcc ctagcaaact ggggcacaaa cttaattgcc
     3181 cgggggatcg gtggagctcc accggtggcg gccgctctag aactagtgag aacctctact
     3241 tccaaggaga tcctctagag tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat
     3301 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     3361 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     3421 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3481 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcact
//
