LOCUS       sequence-342933-        3709 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 3709)
  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 3709)
  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-mScarlet-MCS-polyA
FEATURES             Location/Qualifiers
     source          1..3709
                     /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..1230
                     /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(668..686)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     936..955
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    1512..1528
                     /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(1536..1542,1543..1560,1561..1566))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1581..1602
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1719..1736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1890..2478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1970..1989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2649..3440,3441..3509))
                     /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     3272..3291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3510..3614)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3682..3700
                     /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 aggcagtgat caaggagttc atgcggttca aggtgcacat ggagggctcc
      601 atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
      661 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttctcctg ggacatcctg
      721 tcccctcagt tcatgtacgg ctccagggcc ttcaccaagc accccgccga catccccgac
      781 tactataagc agtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
      841 ggcggcgccg tgaccgtgac ccaggacacc tccctggagg acggcaccct gatctacaag
      901 gtgaagctcc gcggcaccaa cttccctcct gacggccccg taatgcagaa gaagacaatg
      961 ggctgggaag cgtccaccga gcggttgtac cccgaggacg gcgtgctgaa gggcgacatt
     1021 aagatggccc tgcgcctgaa ggacggcggc cgctacctgg cggacttcaa gaccacctac
     1081 aaggccaaga agcccgtgca gatgcccggc gcctacaacg tcgaccgcaa gttggacatc
     1141 acctcccaca acgaggacta caccgtggtg gaacagtacg aacgctccga gggccgccac
     1201 tccaccggcg gcatggacga gctgtacaag taaactagta tcgataagct tgagctcgtc
     1261 gacatcattc tcagtatccc ctgcttatgc actcaccact ttttcccatt attttattca
     1321 tgttgtgttg ctaaattcaa ctacattcgt attaccatgt tgtttcattt gttttttttt
     1381 tatctcagct gtcaaaataa aagcattgaa atacaacatt gttctatttt tcattgtggt
     1441 tttgcatgcc cggtaaataa aaaaacaagg taataaaaat aagatgcgaa atgagaagta
     1501 tatctgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     1561 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     1621 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1681 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     1741 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1801 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1861 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1921 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     1981 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2041 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2101 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2161 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2221 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2281 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2341 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2401 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2461 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2521 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2581 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2641 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2701 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2761 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     2821 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2881 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2941 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3001 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3061 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3121 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3181 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3241 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3301 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3361 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3421 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3481 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3541 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3601 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     3661 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
