LOCUS       sequence-383629-        3530 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Linker::3xFlag::mScarlet::HAtag::Linker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3530)
  AUTHORS   Seleit A, Aulehla A, Paix A
  TITLE     Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 
            knock-in approach.
  JOURNAL   Elife. 2021 Dec 6;10. pii: 75050. doi: 10.7554/eLife.75050.
  PUBMED    34870593
REFERENCE   2  (bases 1 to 3530)
  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: "Elife."; 
            date: "2021-12-6"; volume: "10. pii: 75050. doi"; pages: " 
            10.7554/eLife.75050"
COMMENT     Sequence Label: pAP28
FEATURES             Location/Qualifiers
     source          1..3530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     166..185
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(404..508)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     576..594
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(632..654)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     754..773
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     967..989
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     981..998
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     982..998
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1052..1117
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1118..1810
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(1248..1266)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1516..1535
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1811..1837
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1912..1928)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1912..1928)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1925..1947)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1936..1952
                     /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(1960..1966,1967..1984,1985..1990))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2005..2026
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2143..2160)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2314..2902)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2394..2413)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3073..3530,1..334,335..403))
                     /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"
ORIGIN
        1 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
       61 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
      121 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
      181 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
      241 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
      301 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
      361 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
      421 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
      481 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
      541 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
      601 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
      661 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
      721 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
      781 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
      841 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
      901 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      961 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgagctc ggtacccggg
     1021 gatctctagc ggaccttcag gtagttcgag cgactacaag gaccacgacg gggattataa
     1081 agatcatgat atagattata aagatgatga tgataaagtg agcaagggcg aggcagtgat
     1141 caaggagttc atgcggttca aggtgcacat ggagggctcc atgaacggcc acgagttcga
     1201 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     1261 gaccaagggt ggccccctgc ccttctcctg ggacatcctg tcccctcagt tcatgtacgg
     1321 ctccagggcc ttcaccaagc accccgccga catccccgac tactataagc agtccttccc
     1381 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgccg tgaccgtgac
     1441 ccaggacacc tccctggagg acggcaccct gatctacaag gtgaagctcc gcggcaccaa
     1501 cttccctcct gacggccccg taatgcagaa gaagacaatg ggctgggaag catccaccga
     1561 gcggttgtac cccgaggacg gcgtgctgaa gggcgacatt aagatggccc tgcgcctgaa
     1621 ggacggcggc cgctacctgg cggacttcaa gaccacctac aaggccaaga agcccgtgca
     1681 gatgcccggc gcctacaacg tcgaccgcaa gttggacatc acctcccaca acgaggacta
     1741 caccgtggtg gaacagtacg aacgctccga gggccgccac tccaccggcg gcatggacga
     1801 gctgtacaag tacccatatg atgtcccgga ttacgctagc tctggtccaa gtggatcaag
     1861 ctcggatcct ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct
     1921 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     1981 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2041 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2101 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2161 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2221 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2281 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2341 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2401 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2461 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2521 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2581 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2641 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2701 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2761 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2821 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2881 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2941 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3001 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3061 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3121 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3181 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3241 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3301 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3361 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3421 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3481 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
//
