LOCUS       sequence-348168-        4411 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 4411)
  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 4411)
  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-tdTomato-polyA
FEATURES             Location/Qualifiers
     source          1..4411
                     /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             538..1965
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYE
                     GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMN
                     FEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVL
                     KGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYER
                     SEGRHHLFLYGMDELYK"
     protein_bind    2214..2230
                     /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(2238..2244,2245..2262,2263..2268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2283..2304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2421..2438)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2592..3180)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2672..2691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3351..4142,4143..4211))
                     /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     3974..3993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4212..4316)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4384..4402
                     /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 aggaggtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc
      601 atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
      661 cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg
      721 tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat
      781 tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
      841 ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag
      901 gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg
      961 ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc
     1021 caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac
     1081 atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc
     1141 acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac
     1201 cacctgttcc tggggcatgg caccggcagc accggcagcg gcagctccgg caccgcctcc
     1261 tccgaggaca acaacatggc cgtcatcaaa gagttcatgc gcttcaaggt gcgcatggag
     1321 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     1381 ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac
     1441 atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc
     1501 cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     1561 gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc
     1621 tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag
     1681 accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc
     1741 gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc
     1801 atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg
     1861 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc
     1921 cgccaccacc tgttcctgta cggcatggac gagctgtaca agtaaatcat tctcagtatc
     1981 ccctgcttat gcactcacca ctttttccca ttattttatt catgttgtgt tgctaaattc
     2041 aactacattc gtattaccat gttgtttcat ttgttttttt tttatctcag ctgtcaaaat
     2101 aaaagcattg aaatacaaca ttgttctatt tttcattgtg gttttgcatg cccggtaaat
     2161 aaaaaaacaa ggtaataaaa ataagatgcg aaatgagaag tatatctgtg aaattgttat
     2221 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2281 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2341 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2401 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2461 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2521 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2581 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2641 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2701 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2761 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     2821 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2881 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2941 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3001 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3061 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3121 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3181 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3241 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     3301 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3361 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3421 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3481 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3541 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3601 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3661 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3721 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3781 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3841 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3901 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3961 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4021 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4081 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4141 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4201 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4261 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4321 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     4381 aaaaataggc gtatcacgag gccctttcgt c
//
