LOCUS       sequence-193120-        4384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3XFlag::meGFP with 2 introns::Ollas::H2B::tbb-2 3utr::linker::TEV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4384)
  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 4384)
  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: pAP1071-2
FEATURES             Location/Qualifiers
     source          1..4384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     114..133
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     367..384
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    501..522
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(537..542,543..560,561..567)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    575..591
                     /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)."
     primer_bind     580..602
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     599..615
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     599..615
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(663..683)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1433..1474)
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     primer_bind     complement(1515..1535)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     2232..2260
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             complement(2291..2356)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2383..2400)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2383..2399)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2392..2414)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2608..2627)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2727..2749
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2787..2805)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2873..2977
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2978..3046,3047..3838)
                     /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     complement(3196..3215)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4009..4384,1..213)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
       61 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
      121 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
      181 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
      241 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
      301 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
      361 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
      421 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
      481 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
      541 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
      601 ggaaacagct atgaccatga ttacgccaag cttgcatgcc tgcaggtcga ctctagagga
      661 tctccttgga agtagaggtt ctcactagtt ctagagcggc cgccaccggt ggagctccac
      721 cgatcccccg ggcatttctt ggcggcacaa taaagtttcc tctttttgtt gggtcactca
      781 acgaacacaa tacaaatatt tgagcgctcg catttttctc aacctttcgg gtttaaagca
      841 cttcaaaatg gaattgaaga gagattgtga aagtacatca gacagaatca cattttgata
      901 gcattcactt cactcagatg caaacttttc agtgactata aaaagtcata aaataataac
      961 attaaaaaag caaataaatt agcgagagaa ttttttgaca aaaagaaaga agagtgatag
     1021 agaagaaggg aatgcttgaa aggatcttgc atttatcact agtttacttg ctggaagtgt
     1081 acttggtgac ggccttggtt ccctcagaca cggcgtgctt ggcaagttct cctgggagaa
     1141 tcaaacggac agcggtttga atttcgcggg atgagatcgt tgagcgtttg ttgtaatgag
     1201 caagacggga agcttccgaa gcgatgcgtt cgaatacatc gttgacgaag gagttcatga
     1261 tagacatggc cttggaggag actccggtgt ctgggtgaac ttgcttgaga acacggtaga
     1321 tgtagacgga gtacgattcc ttgcgggcat gacgtctctt ctttccgtcc tttggcttgg
     1381 caacgacggt cttggcggcc ttcttggctc ccttggcaga tggctttggt ggctttccca
     1441 tgagacgtgg tccgagctcg ttggcgaatc cggacttgta cagctcgtcc atgccgagtg
     1501 taatcccagc agctgttaca aactcaagaa ggaccatgtg gtctctcttt tcgttgggat
     1561 ctttcgaaag cttagattgt gtggacaggt aatggttgtc tggtaaaagg acagggccat
     1621 cgccaattgg agtattttgt tgataatggt ctgctagttg aacgcttcca tcttcaatgt
     1681 tgtgtctaat tttgaagtta actttgattc cattctgaaa atttaaatat gtatggttag
     1741 ttagtaccga actgtttaaa cttacctttt gtttgtctgc catgatgtat acattgtgtg
     1801 agttatagtt gtattccaat ttgtgtccaa gaatgtttcc atcttcttta aaatcaatac
     1861 cttttaactc gattctatta acaagggtat caccttcaaa cttgacttca gcacgtgtct
     1921 tgtagttccc gtcatctttg aaaaatatag ttctttcctg tacataacct tcgggcatgg
     1981 cactctgaaa atttaaataa tcagggttag ttagtatata tatgtttaaa cttaccttga
     2041 aaaagtcatg ctgcttcatg tggtcggggt accggctgaa gcactgcacg ccgtaggtca
     2101 gagtagtgac aagtgttggc catggaacag gtagttttcc agtagtgcaa ataaatttaa
     2161 gggtaagttt tccgtatgtt gcatcacctt caccctctcc actgacagaa aatttgtgcc
     2221 cattaacatc accatctaat tcaacaagaa ttgggacaac tccagtgaaa agttcttctc
     2281 ctttactcac cttgtcatcg tcatccttgt aatcgatatc atgatcttta taatcaccgt
     2341 catggtcttt gtagtcgatc cccgggtacc gagctcgaat tcactggccg tcgttttaca
     2401 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2461 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     2521 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2581 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2641 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     2701 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     2761 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2821 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2881 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2941 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3001 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3061 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3121 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3181 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3241 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3301 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3361 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3421 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3481 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     3541 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3601 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3661 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3721 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3781 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3841 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3901 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3961 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4021 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4081 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4141 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4201 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4261 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4321 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4381 gaga
//
