LOCUS       sequence-339214-        5008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for synthesis of mRNA for injection into xenopus laevis embryos.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5008)
  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 5008)
  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 Bighead S-HA
FEATURES             Location/Qualifiers
     source          1..5008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    74..98
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(928..952)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             960..986
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1026..1160
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1050..1069)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1104..1123
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1273..1293)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1273..1291)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1312..1328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1312..1328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1325..1347)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1336..1352
                     /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(1360..1366,1367..1384,1385..1390))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1405..1426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1543..1560)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1714..2302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1794..1813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2473..3264,3265..3333))
                     /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     3096..3115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3334..3438)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3464..3919)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3601..3622)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3813..3832
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3990..4974
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 atttaggtga cactatagaa tacaagctac ttgttctttt tgcaggatcc catcgattcg
       61 aattcaagga tcaacaagtt tgtacaaaaa agcaggctac catgggtaca gtttggctct
      121 ggatatgcct agttttacag gtttcttgga ctttccccat gcactttagg aaacataatg
      181 aactcacatt gctgagaaac aaagtggaaa gccatggaga tcccaataat ttcatcaaac
      241 aaagcagagc agatactccc tttaaggaaa gagtgggcac cttcccggag atgactggtg
      301 ggagacgtag caacagacag aacatactga aatactatcg tgcgcagtgt acctacatga
      361 gccaaccatg gatacagagc caggatatta tttaccaaca ggaccccagc gccatgttcc
      421 tgttcaaagt ctatacaggg tccttgaggc caacttttcc tcatcagaat cttttcaaat
      481 acatgagcag aatttatcgg tgctgcaaac ttgggtttac ctgtaagagg gtaaaaggcc
      541 ttcagggaac ctggactcct ggtagtggga agaaagaggt ggaattctcc attgactccg
      601 acatctttaa tctttcgatt ctaagggctg aactccactt ggaagtgtcc cttgcaagta
      661 accagctcac agttgttccg ggccttacag caaaaggcat cacactcgca agctttacag
      721 aattaaggag tgatcatgtc atggaactgg cactggatgc aatgtccctt ttgcaaatgt
      781 taaaagaatc agatacagag gatacaaatg aagatgtaac agagctgagc ttggcattgc
      841 actgcataca caaggagcac cctgttcctt gccatctcta tggggtgtca ctgcttcacc
      901 caccctttct agcttttcag tacgaagacc cagctttctt gtacaaagtg gttgatcctt
      961 acccatacga cgtcccagac tacgcttaat cgagcctcta gaactatagt gagtcgtatt
     1021 acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     1081 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     1141 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     1201 gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc
     1261 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     1321 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccgggagcat
     1381 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     1441 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1501 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     1561 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1621 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     1681 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1741 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1801 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1861 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1921 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1981 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2041 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2101 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2161 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2221 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2281 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2341 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2401 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2461 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2521 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2581 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     2641 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2701 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2761 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2821 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2881 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2941 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3001 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3061 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3121 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3181 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3241 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3301 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3361 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3421 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     3481 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     3541 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     3601 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     3661 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     3721 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     3781 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     3841 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     3901 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     3961 tcggtgcggg cctcttcgct attacgccag tcgaccgcca attcaatatg gcgtatatgg
     4021 actcatgcca attcaatatg gtggatctgg acctgtgcca attcaatatg gcgtatatgg
     4081 actcgtgcca attcaatatg gtggatctgg accccagcca attcaatatg gcggacttgg
     4141 caccatgcca attcaatatg gcggacctgg cactgtgcca actggggagg ggtctacttg
     4201 gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc aagtccgcca tattgaattg
     4261 gcatggtgcc aataatggcg gccatattgg ctatatgcca ggatcaatat ataggcaata
     4321 tccaatatgg ccctatgcca atatggctat tggccaggtt caatactatg tattggccct
     4381 atgccatata gtattccata tatgggtttt cctattgacg tagatagccc ctcccaatgg
     4441 gcggtcccat ataccatata tggggcttcc taataccgcc catagccact cccccattga
     4501 cgtcaatggt ctctatatat ggtctttcct attgacgtca tatgggcggt cctattgacg
     4561 tatatggcgc ctcccccatt gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc
     4621 attgacgtca ataggaccac ccaccattga cgtcaatggg atggctcatt gcccattcat
     4681 atccgttctc acgcccccta ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat
     4741 caatatctat taatagtaac ttggcaagta cattactatt ggaagtacgc cagggtacat
     4801 tggcagtact cccattgacg tcaatggcgg taaatggccc gcgatggctg ccaagtacat
     4861 ccccattgac gtcaatgggg aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg
     4921 ggcggtaggc gtgcctaatg ggaggtctat ataagcaatg ctcgtttagg gaaccgccat
     4981 tctgcctggg gacgtcggag caagcttg
//
