LOCUS       sequence-339218-        5505 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 5505)
  AUTHORS   Ding Y, Colozza G, Zhang K, Moriyama Y, Ploper D, Sosa EA, Benitez 
            MDJ, De Robertis EM
  TITLE     Genome-wide analysis of dorsal and ventral transcriptomes of the 
            Xenopus laevis gastrula.
  JOURNAL   Dev Biol. 2017 Jun 15;426(2):176-187. doi: 
            10.1016/j.ydbio.2016.02.032. Epub 2016 Mar 23.
  PUBMED    27016259
REFERENCE   2  (bases 1 to 5505)
  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: 
            "10.1016/j.ydbio.2016.02.032"; journalName: "Dev Biol"; date: 
            "2017-06-15- 15"; volume: "426"; issue: "2"; pages: "176-187"
COMMENT     Sequence Label: pCS2xVlk1-3xFlag
FEATURES             Location/Qualifiers
     source          1..5505
                     /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"
     CDS             1411..1482
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     polyA_signal    1523..1657
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1547..1566)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1601..1620
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1770..1790)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1770..1788)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1809..1825)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1809..1825)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1822..1844)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1833..1849
                     /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(1857..1863,1864..1881,1882..1887))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1902..1923
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2040..2057)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2211..2799)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2291..2310)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2970..3761,3762..3830))
                     /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     3593..3612
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3831..3935)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3961..4416)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4098..4119)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4310..4329
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4487..5471
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 atttaggtga cactatagaa tacaagctac ttgttctttt tgcaggatcc catcgatatg
       61 aagaacacca ttatcctgtg cggagctgca gtctctgtgc tgtctgtgtt ttctctgctc
      121 ttcccccgac ccccgagccg caaatcattt gtgagatctg agcgccaggc cgagctgcac
      181 agggaaatcc tggagagtcg ccgggatctg ctcttgtttc tctcttctct tctggagggg
      241 tcgggggcag atgagttgct gctgcccggg gacataatgg gtcggacagg gactttatta
      301 ggctgcgagg atctcacgta tgtaactggc ctccagtacg tgggctccgg attcactaaa
      361 cttgttcaca agggaacttt gcgcaacggg aaggagatcg ctctcaagtc tgtgcacaat
      421 gagggcaacg atgtgactcg ctgcgtcaga cgctacggcg accccgctgg ctgccgctct
      481 ctcgccactt acaaactcca caaggaggtc actctgctcc ggaccttgcg ccacccgggc
      541 gtcattacgt tgcatggaca ttgctacgag aatagcctgg ctcctaactt tagggtcaca
      601 gctatgctgg aactgggatc tcccctagag atgattcagc ttctgcagac cccctgggag
      661 aagagattta aaatttgcct agaactggta aatttactgc attatctagc caactctccc
      721 atgggatcta ttgccctcct ggattttcag ccaaggcagt ttgtcctggt tgatgaaaac
      781 ctgaaagtaa cagatataga tgatgccaca atggaggagc ttgagtgcaa cacggacaag
      841 gattgtactc ttgaatttcc atccaggaca tttgctgtca ggtgctcccg tgctgggaga
      901 tgcactggca tcaatgaaaa aaggaacctc ttcaatgcat acaggttttt cttcacatat
      961 ctgctcccgc acgcagcgcc tcctgctttg cagcctcttc tcttggacat tatgaatgcc
     1021 acaggggact tgagctatgg cattaatgag accctggaag ctttccagaa tgttttgaag
     1081 ctgtacaaat cgggaatgaa ccatcagagg aggctgctat atttgaaaga ctacatgctg
     1141 atagaaggat acagactaaa tgaaaccaat gacgacttca gatgttggcc ctcctacaat
     1201 cacagagggt gcgcgctgtc tgtgcacaat tcagaagagg ctgccgagtt ctgtagcaag
     1261 aatccaaact gtcaccactt tgtcattggt catcaaagaa catggacagg ccgcctgatg
     1321 gcaacattca ccagtagtgc ccacaaactt atccctgatg cgaattcccg tgtttacatg
     1381 aaaaaaccaa gcaacctcaa aaagaggcct gattataaag atgacgatga caaggattat
     1441 aaagatgacg atgacaagga ttataaagat gacgatgaca agtaactcga gcctctagaa
     1501 ctatagtgag tcgtattacg tagatccaga catgataaga tacattgatg agtttggaca
     1561 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     1621 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     1681 tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcgcggccg cggcgccaat
     1741 gcattgggcc cggtacccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     1801 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     1861 atacgagccg ggagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     1921 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     1981 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2041 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2101 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2161 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2221 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2281 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2341 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2401 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2461 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2521 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2581 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2641 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2701 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2761 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2821 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2881 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2941 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3001 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3061 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3121 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3181 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3241 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3301 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3361 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3421 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3481 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3541 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3601 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3661 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3721 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3781 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3841 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3901 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3961 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     4021 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     4081 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     4141 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     4201 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     4261 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     4321 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     4381 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
     4441 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagtcg accgccaatt
     4501 caatatggcg tatatggact catgccaatt caatatggtg gatctggacc tgtgccaatt
     4561 caatatggcg tatatggact cgtgccaatt caatatggtg gatctggacc ccagccaatt
     4621 caatatggcg gacttggcac catgccaatt caatatggcg gacctggcac tgtgccaact
     4681 ggggaggggt ctacttggca cggtgccaag tttgaggagg ggtcttggcc ctgtgccaag
     4741 tccgccatat tgaattggca tggtgccaat aatggcggcc atattggcta tatgccagga
     4801 tcaatatata ggcaatatcc aatatggccc tatgccaata tggctattgg ccaggttcaa
     4861 tactatgtat tggccctatg ccatatagta ttccatatat gggttttcct attgacgtag
     4921 atagcccctc ccaatgggcg gtcccatata ccatatatgg ggcttcctaa taccgcccat
     4981 agccactccc ccattgacgt caatggtctc tatatatggt ctttcctatt gacgtcatat
     5041 gggcggtcct attgacgtat atggcgcctc ccccattgac gtcaattacg gtaaatggcc
     5101 cgcctggctc aatgcccatt gacgtcaata ggaccaccca ccattgacgt caatgggatg
     5161 gctcattgcc cattcatatc cgttctcacg ccccctattg acgtcaatga cggtaaatgg
     5221 cccacttggc agtacatcaa tatctattaa tagtaacttg gcaagtacat tactattgga
     5281 agtacgccag ggtacattgg cagtactccc attgacgtca atggcggtaa atggcccgcg
     5341 atggctgcca agtacatccc cattgacgtc aatggggagg ggcaatgacg caaatgggcg
     5401 ttccattgac gtaaatgggc ggtaggcgtg cctaatggga ggtctatata agcaatgctc
     5461 gtttagggaa ccgccattct gcctggggac gtcggagcaa gcttg
//
