LOCUS       sequence-383628-        3542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Linker::3xFlag::mNeonGreen::HAtag::Linker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3542)
  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 3542)
  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: pAP27
FEATURES             Location/Qualifiers
     source          1..3542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     207..226
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(445..549)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     617..635
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(673..695)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     795..814
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1008..1030
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1022..1039
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1023..1039
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1093..1158
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1864..1890
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1965..1981)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1965..1981)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1978..2000)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1989..2005
                     /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(2013..2019,2020..2037,2038..2043))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2058..2079
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2196..2213)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2367..2955)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2447..2466)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3126..3542,1..375,376..444))
                     /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 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
       61 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
      121 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
      181 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
      241 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
      301 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
      361 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
      421 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
      481 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
      541 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
      601 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
      661 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
      721 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
      781 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
      841 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct
      901 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      961 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     1021 ttgtaaaacg acggccagtg aattcgagct cggtacccgg ggatctctag cggaccttca
     1081 ggtagttcga gcgactacaa ggaccacgac ggggattata aagatcatga tatagattat
     1141 aaagatgatg atgataaagt ctccaaaggg gaggaagaca acatggcgtc cctcccagcg
     1201 acgcacgaac tccatatctt tgggtccata aacggtgtcg atttcgacat ggtaggacaa
     1261 gggaccggaa atccaaatga tggatatgag gagctaaacc tcaagagtac caagggggac
     1321 ctccaatttt ccccatggat actcgtcccc catatcgggt acggattcca tcaataccta
     1381 ccatatccag atgggatgtc cccatttcaa gcggccatgg tagacggaag tggataccaa
     1441 gtacatcgta cgatgcaatt tgaggacggg gcctccctaa ccgtcaacta tcgttacacg
     1501 tacgagggaa gtcacatcaa aggagaggcg caagtcaaag gaactggatt tccagccgat
     1561 ggaccagtca tgacgaattc cctaaccgcc gccgattggt gtcgtagtaa aaagacgtac
     1621 ccaaatgaca agaccataat ctccacgttc aagtggtcct ataccaccgg gaacggaaag
     1681 cggtaccgta gtacggcccg gacgacgtac acgttcgcca agcccatggc cgctaattac
     1741 ctgaagaatc agccaatgta tgtcttccgt aaaaccgagc taaagcactc caaaaccgag
     1801 ctaaacttca aggaatggca aaaggcgttc acggacgtaa tgggaatgga cgaactctac
     1861 aagtacccat atgatgtccc ggattacgct agctctggtc caagtggatc aagctcggat
     1921 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
     1981 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     2041 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     2101 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2161 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2221 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2281 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2341 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2401 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2461 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2521 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2581 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2641 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2701 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2761 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     2821 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2881 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2941 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3001 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3061 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3121 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3181 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3241 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3301 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3361 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3421 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3481 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3541 aa
//
