LOCUS       sequence-339212-        4817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mRNA synthesis for injection into embryos.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4817)
  AUTHORS   Ding Y, Colozza G, Sosa EA, Moriyama Y, Rundle S, Salwinski L, De 
            Robertis EM
  TITLE     Bighead is a Wnt antagonist secreted by the Xenopus Spemann 
            organizer that promotes Lrp6 endocytosis.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Sep 25;115(39):E9135-E9144. doi: 
            10.1073/pnas.1812117115. Epub 2018 Sep 12.
  PUBMED    30209221
REFERENCE   2  (bases 1 to 4817)
  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"; doi: 
            "10.1073/pnas.1812117115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-09-25- 25"; volume: "115"; issue: "39"; pages: 
            "E9135-E9144"
COMMENT     Sequence Label: pCS2-xFAM3b.L-HA
FEATURES             Location/Qualifiers
     source          1..4817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        372..390
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     372..389
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1140..1166
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1206..1340
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1230..1249)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1284..1303
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1453..1473)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1453..1471)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1492..1508)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1492..1508)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1505..1527)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1516..1532
                     /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(1540..1546,1547..1564,1565..1570))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1585..1606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1723..1740)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1894..2482)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1974..1993)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2653..3444,3445..3513))
                     /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     3276..3295
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3514..3618)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3644..4099)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3781..3802)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3993..4012
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(4170..4817,1..337)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cacccaccat tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc
       61 ctattgacgt caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt
      121 aacttggcaa gtacattact attggaagta cgccagggta cattggcagt actcccattg
      181 acgtcaatgg cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg
      241 gggaggggca atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta
      301 atgggaggtc tatataagca atgctcgttt agggaaccgc cattctgcct ggggacgtcg
      361 gagcaagctt gatttaggtg acactataga atacaagcta cttgttcttt ttgcaggatc
      421 ccatcgatac catggtcaac ttcaggttcc tgtcgagcaa ctccgtgagg gtggtgggcc
      481 tgctatttgc ctctgtgtgt gcctggtaca tgggatacct gtttgcagag gtattgccag
      541 aagaaccctg gcagtcggca gttggtagca tgcaaagcat tggccagaga ccggttctaa
      601 aagctcccaa tcccaaaagg cagaaatgtg acatctgggt gtcctgtgca ccaaaccaac
      661 ttgcctaccg catacggact ggcggcgcaa aagacgtcat gcccgacatt tgtttagatg
      721 ataacatact cctctcggga aagaattcca accgagggat aaacatcgct gttgttagtg
      781 ccgaaacctg gaagctcgtt gagaccaaaa catttgacat gtatgaagga gatttttccg
      841 gcgctatggt tgaattcctt aacaagatac cgaatggggc tctcattatg gtcgccagtc
      901 atgatgatgc ctttaccaag ttgactgaac ccgcaaaaaa agccttcgaa gacttcggga
      961 gcaaagaagt ccggaacctt cgcttccgat cagcttgggc ctttttggcc attaaaggaa
     1021 ctaaaacacc cgacaacttt gaaaaagaaa agatcaacca ctcggacgga agcaagaaca
     1081 gatattccgg ttggccggcg gaaattcaga tcgacggatg tctcccaaaa gcaaggcctt
     1141 acccatacga cgtcccagac tacgcttaat cgagcctcta gaactatagt gagtcgtatt
     1201 acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     1261 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     1321 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     1381 gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc
     1441 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     1501 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccgggagcat
     1561 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     1621 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1681 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     1741 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1801 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     1861 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1921 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1981 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2041 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2101 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2161 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2221 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2281 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2341 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2401 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2461 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2521 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2581 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2641 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2701 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2761 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     2821 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2881 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2941 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3001 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3061 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3121 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3181 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3241 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3301 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3361 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3421 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3481 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3541 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3601 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     3661 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     3721 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     3781 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     3841 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     3901 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     3961 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     4021 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     4081 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     4141 tcggtgcggg cctcttcgct attacgccag tcgaccgcca attcaatatg gcgtatatgg
     4201 actcatgcca attcaatatg gtggatctgg acctgtgcca attcaatatg gcgtatatgg
     4261 actcgtgcca attcaatatg gtggatctgg accccagcca attcaatatg gcggacttgg
     4321 caccatgcca attcaatatg gcggacctgg cactgtgcca actggggagg ggtctacttg
     4381 gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc aagtccgcca tattgaattg
     4441 gcatggtgcc aataatggcg gccatattgg ctatatgcca ggatcaatat ataggcaata
     4501 tccaatatgg ccctatgcca atatggctat tggccaggtt caatactatg tattggccct
     4561 atgccatata gtattccata tatgggtttt cctattgacg tagatagccc ctcccaatgg
     4621 gcggtcccat ataccatata tggggcttcc taataccgcc catagccact cccccattga
     4681 cgtcaatggt ctctatatat ggtctttcct attgacgtca tatgggcggt cctattgacg
     4741 tatatggcgc ctcccccatt gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc
     4801 attgacgtca ataggac
//
