LOCUS       sequence-193533-        3536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEV::linker::tagRFP::3XFlag.
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: pAP974-1
FEATURES             Location/Qualifiers
     source          1..3536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(110..901,902..970))
                     /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     733..752
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(971..1075)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1143..1161
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1199..1221)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1321..1340
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1534..1556
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1548..1565
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1549..1565
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1592..1612
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1664..2371
                     /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             2372..2437
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2485..2501)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2485..2501)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2498..2520)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2509..2525
                     /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(2533..2539,2540..2557,2558..2563))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2578..2599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2716..2733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2887..3475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2967..2986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
       61 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
      121 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
      181 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
      241 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
      301 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
      361 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
      421 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
      481 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
      541 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
      601 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
      661 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
      721 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
      781 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
      841 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
      901 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
      961 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     1021 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     1081 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     1141 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     1201 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     1261 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
     1321 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
     1381 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg
     1441 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     1501 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     1561 ccagtgaatt cgagctcggt acccggggat cgagaacctc tacttccaag gatgcccggg
     1621 ggatcggtgg agctccaccg gtggcggccg ctctagaact agtgtgtcta agggcgaaga
     1681 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtga acaaccacca
     1741 cttcaaatgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat
     1801 caaggtggtc gagggcggcc ctctcccctt cgcctttgac atcctggcta ccagcttcat
     1861 gtacggcagc agaaccttca tcaaccacac ccagggcatc cccgacttct ttaagcagtc
     1921 ctttcctgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac
     1981 cgctacccag gacaccagcc tccaggacgg ctgtctcatc tacaacgtca agatcagagg
     2041 ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa acactcggct gggaggccaa
     2101 taccgagatg ctgtaccccg ctgacggcgg cctggaaggc agaagcgaca tggccctgaa
     2161 gctcgtgggc gggggccacc tgatctgcaa tttcaagacc acatacagat ccaagaaacc
     2221 cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac cacagactgg aaagaatcaa
     2281 agaggccgac aaagagacct acgtcgagca gcacgaggtg gctgtggcca gatactgcga
     2341 cctccctagc aaactggggc acaaacttaa tgactacaaa gaccatgacg gtgattataa
     2401 agatcatgat atcgattaca aggatgacga tgacaaggat cctctagagt cgacctgcag
     2461 gcatgcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     2521 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     2581 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2641 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2701 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2761 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2821 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2881 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2941 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3001 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3061 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3121 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3181 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3241 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3301 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3361 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3421 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3481 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaag
//
