LOCUS       sequence-342934-        3853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid is used for generating C-terminal knock-in plasmid 
            and/or PCR donors in zebrafish..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3853)
  AUTHORS   Levic DS, Yamaguchi N, Wang S, Knaut H, Bagnat M
  TITLE     Knock-in tagging in zebrafish facilitated by insertion into 
            non-coding regions.
  JOURNAL   Development. 2021 Sep 8. pii: 272090. doi: 10.1242/dev.199994.
  PUBMED    34495314
REFERENCE   2  (bases 1 to 3853)
  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: 
            "Development. 2021 Sep 8. pii: 272090. doi: 10.1242/dev.199994."
COMMENT     Sequence Label: pUC19-TgKI-MCS-p2A-Venus-PEST-MCS-polyA
FEATURES             Location/Qualifiers
     source          1..3853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             481..537
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(538..540,541..1251,1252..1254,1255..1377)
                     /codon_start=1
                     /product="EYFP destabilized by residues 422-461 of mouse 
                     ornithine decarboxylase, giving an in vivo half-life of ~2 
                     hours"
                     /label=d2EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYKLSHGFPPAVAAQDDGTLPMSCAQESGMDRHPAACASARINV"
     primer_bind     complement(580..601)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(841..860)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1188..1209
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1656..1672
                     /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(1680..1686,1687..1704,1705..1710))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1725..1746
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1863..1880)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2034..2622)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2114..2133)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2793..3584,3585..3653))
                     /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     3416..3435
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3654..3758)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3826..3844
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtggcga gggcgatgcc acctacggga
      421 tgcggccgca ccggtggatc cgaattcgct agcccgcggt ctagactcga gggatccgga
      481 gccacgaact tctctctgtt aaagcaagca ggagacgtgg aagaaaaccc cggtcctgtg
      541 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      601 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      661 ctgaccctga agctgatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      721 accaccctgg gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
      781 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
      841 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
      901 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
      961 gagtacaact acaacagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc
     1021 aaggccaact tcaagatccg ccacaacatc gaggacggcg gcgtgcagct cgccgaccac
     1081 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1141 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1201 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gcttagccat
     1261 ggcttcccgc cggcggtggc ggcgcaggat gatggcacgc tgcccatgtc ttgtgcccag
     1321 gagagcggga tggaccgtca ccctgcagcc tgtgcttctg ctaggatcaa tgtgtagact
     1381 agtatcgata agcttgagct cgtcgacatc attctcagta tcccctgctt atgcactcac
     1441 cactttttcc cattatttta ttcatgttgt gttgctaaat tcaactacat tcgtattacc
     1501 atgttgtttc atttgttttt tttttatctc agctgtcaaa ataaaagcat tgaaatacaa
     1561 cattgttcta tttttcattg tggttttgca tgcccggtaa ataaaaaaac aaggtaataa
     1621 aaataagatg cgaaatgaga agtatatctg tgaaattgtt atccgctcac aattccacac
     1681 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     1741 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1801 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1861 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1921 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1981 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2041 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2101 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2161 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2221 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2281 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2341 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2401 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2461 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2521 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2581 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2641 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2701 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2761 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2821 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2881 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2941 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3001 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3061 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3121 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3181 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3241 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3301 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3361 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3421 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3481 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3541 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3601 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3661 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3721 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3781 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     3841 aggccctttc gtc
//
