LOCUS       sequence-342932-        3730 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 3730)
  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 3730)
  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-eGFP-MCS-polyA
FEATURES             Location/Qualifiers
     source          1..3730
                     /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..1254)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(538..540,541..1251)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     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    1533..1549
                     /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(1557..1563,1564..1581,1582..1587))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1602..1623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1740..1757)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1911..2499)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1991..2010)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2670..3461,3462..3530))
                     /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     3293..3312
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3531..3635)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3703..3721
                     /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 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      721 accaccctga cctacggcgt gcagtgcttc agccgctacc 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 atcatggccg acaagcagaa gaacggcatc
     1021 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1081 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1141 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1201 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaactagt
     1261 atcgataagc ttgagctcgt cgacatcatt ctcagtatcc cctgcttatg cactcaccac
     1321 tttttcccat tattttattc atgttgtgtt gctaaattca actacattcg tattaccatg
     1381 ttgtttcatt tgtttttttt ttatctcagc tgtcaaaata aaagcattga aatacaacat
     1441 tgttctattt ttcattgtgg ttttgcatgc ccggtaaata aaaaaacaag gtaataaaaa
     1501 taagatgcga aatgagaagt atatctgtga aattgttatc cgctcacaat tccacacaac
     1561 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     1621 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     1681 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     1741 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     1801 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1861 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1921 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1981 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2041 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2101 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2161 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2221 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2281 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2341 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2401 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2461 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2521 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2581 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2641 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     2701 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2761 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2821 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     2881 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     2941 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3001 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3061 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3121 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3181 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3241 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3301 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3361 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3421 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3481 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3541 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3601 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3661 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     3721 ccctttcgtc
//
