LOCUS       sequence-358186-        5655 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 5655)
  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 5655)
  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-FC
FEATURES             Location/Qualifiers
     source          1..5655
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        113..1097
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1132..1150
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1132..1149
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    1205..1229
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2059..2083)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2770..2789)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2771..2789)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2804..2938
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2828..2847)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2882..2901
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3051..3071)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3051..3069)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3090..3106)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3090..3106)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3103..3125)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3114..3130
                     /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(3138..3144,3145..3162,3163..3168))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3183..3204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3321..3338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3492..4080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3572..3591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4251..5042,5043..5111))
                     /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     4874..4893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5112..5216)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5242..5655,1..42))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5379..5400)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5591..5610
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
       61 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagtcgaccg
      121 ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc tggacctgtg
      181 ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc tggaccccag
      241 ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggacc tggcactgtg
      301 ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc ttggccctgt
      361 gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat tggctatatg
      421 ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc tattggccag
      481 gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt tttcctattg
      541 acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct tcctaatacc
      601 gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt cctattgacg
      661 tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca attacggtaa
      721 atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat tgacgtcaat
      781 gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt caatgacggt
      841 aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa gtacattact
      901 attggaagta cgccagggta cattggcagt actcccattg acgtcaatgg cggtaaatgg
      961 cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca atgacgcaaa
     1021 tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc tatataagca
     1081 atgctcgttt agggaaccgc cattctgcct ggggacgtcg gagcaagctt gatttaggtg
     1141 acactataga atacaagcta cttgttcttt ttgcaggatc ccatcgattc gaattcaagg
     1201 atcaacaagt ttgtacaaaa aagcaggcta ccatgggtac agtttggctc tggatatgcc
     1261 tagttttaca ggtttcttgg actttcccca tgcactttag gaaacataat gaactcacat
     1321 tgctgagaaa caaagtggaa agccatggag atcccaataa tttcatcaaa caaagcagag
     1381 cagatactcc ctttaaggaa agagtgggca ccttcccgga gatgactggt gggagacgta
     1441 gcaacagaca gaacatactg aaatactatc gtgcgcagtg tacctacatg agccaaccat
     1501 ggatacagag ccaggatatt atttaccaac aggaccccag cgccatgttc ctgttcaaag
     1561 tctatacagg gtccttgagg ccaacttttc ctcatcagaa tcttttcaaa tacatgagca
     1621 gaatttatcg gtgctgcaaa cttgggttta cctgtaagag ggtaaaaggc cttcagggaa
     1681 cctggactcc tggtagtggg aagaaagagg tggaattctc cattgactcc gacatcttta
     1741 atctttcgat tctaagggct gaactccact tggaagtgtc ccttgcaagt aaccagctca
     1801 cagttgttcc gggccttaca gcaaaaggca tcacactcgc aagctttaca gaattaagga
     1861 gtgatcatgt catggaactg gcactggatg caatgtccct tttgcaaatg ttaaaagaat
     1921 cagatacaga ggatacaaat gaagatgtaa cagagctgag cttggcattg cactgcatac
     1981 acaaggagca ccctgttcct tgccatctct atggggtgtc actgcttcac ccaccctttc
     2041 tagcttttca gtacgaagac ccagctttct tgtacaaagt ggttgatcct ctcgagccac
     2101 ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag gacaccctca
     2161 tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg
     2221 aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag acaaagccgc
     2281 gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg
     2341 actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc ccagccccca
     2401 tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg tacaccctgc
     2461 ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg gtcaaaggct
     2521 tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag aacaactaca
     2581 agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc aagctcaccg
     2641 tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg catgaggctc
     2701 tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga tctagaggta
     2761 cccaattcgc cctatagtga gtcgtattac aattcactac gtagatccag acatgataag
     2821 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     2881 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     2941 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     3001 attcgcggcc gcggcgccaa tgcattgggc ccggtaccca gcttttgttc cctttagtga
     3061 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3121 ccgctcacaa ttccacacaa catacgagcc gggagcataa agtgtaaagc ctggggtgcc
     3181 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3241 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3301 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3361 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3421 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3481 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3541 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3601 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3661 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3721 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3781 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3841 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3901 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3961 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4021 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4081 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4141 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4201 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4261 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4321 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4381 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4441 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4501 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4561 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4621 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4681 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4741 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4801 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4861 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4921 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4981 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5041 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5101 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5161 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5221 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     5281 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     5341 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     5401 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     5461 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     5521 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     5581 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     5641 tcacgctgcg cgtaa
//
