LOCUS       sequence-461338-        7434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Optogenetic Nodal activators from Sako et al. 2016 (Cell Reports) 
            separated by a P2A peptide for singe-transcript expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7434)
  AUTHORS   Iannucci LE, Thomas VSM, Murphy MR, Donahue CET, Anderson WK, Rogers
            CE, Saul AJ, Rogers KW
  TITLE     
  JOURNAL   bioRxiv [Preprint]. 2025 Apr 19:2025.04.17.649426. doi: 
            10.1101/2025.04.17.649426.
  PUBMED    40568079
REFERENCE   2  (bases 1 to 7434)
  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: "bioRxiv 
            [Preprint]. 2025 Apr 19:2025.04.17.649426. doi: 
            10.1101/2025.04.17.649426."
COMMENT     Sequence Label: pCS2-Opto-Acvr1ba-P2A-Acvr2ba
FEATURES             Location/Qualifiers
     source          1..7434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      85..94
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             91..132
                     /codon_start=1
                     /product="N-myristoylation signal from Src kinase (Pellman 
                     et al., 1985; Kaplan et al., 1988)"
                     /label=myr
                     /translation="MGSSKSKPKDPSQR"
     CDS             1714..1740
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1750..1806
                     /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             1807..1848
                     /codon_start=1
                     /product="N-myristoylation signal from Src kinase (Pellman 
                     et al., 1985; Kaplan et al., 1988)"
                     /label=myr
                     /translation="MGSSKSKPKDPSQR"
     CDS             3430..3453
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3486..3620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3510..3529)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3564..3583
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3733..3753)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3733..3751)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3772..3788)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3772..3788)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3785..3807)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3796..3812
                     /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(3820..3826,3827..3844,3845..3850))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3865..3886
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4003..4020)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4174..4762)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4254..4273)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4933..5724,5725..5793))
                     /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     5556..5575
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5794..5898)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5924..6379)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6061..6082)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6273..6292
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6450..7434
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atccgccacc atggggagta gcaagagcaa gcctaaggac
      121 cccagccagc gcggtggagg aggttctgga ggcggtggaa gtggtggcgg aggtagccac
      181 cggcagcgac tggatgttga ggatccatcc tgtgatcacc tgtacttggc caaagacaag
      241 accttacagg atctcatctt cgatctgtcc acctctggtt cagggtctgg gctgcccttg
      301 ttcgttcaga ggactgtggc caggacaatt gtactgcagg agatcatagg aaaaggtcgc
      361 tttggggaag tgtggagggg gagatggaga ggaggtgatg tggctgtgaa gatcttctca
      421 tccagggagg aacgttcctg gttccgtgaa gctgagattt accaaaccat catgctccgc
      481 catgaaaaca tcttgggctt cattgctgct gataataaag acaatggcac atggacacag
      541 ctgtggctag tgtcagacta ccatgagcat ggctcattat ttgactatct caaccactac
      601 tccgtcacaa tcgaggggat gatcaagtta tcgctttcag ccgccagcgg tctggcacat
      661 ctgcacatgg agatcctggg cacacagggg aaaccaggca tcgcacatcg tgacctcaaa
      721 tccaaaaaca tcttggtgaa aaagaatggt acttgcgcca tagcagatct agggctcgct
      781 gtacggcacg agtccatcac tgatactata gacattgcac ccaatcagag ggtgggcact
      841 aaaagatata tggccccaga ggtactagat gaaacgatca acatgaagca ttttgattct
      901 ttcaagtgtg ctgatatcta tgctttggga ctggtatatt gggagattgc acgacggtgt
      961 aatgctggag gtatccatga agattatcag ctaccctact atgacctggt gccgtccgac
     1021 ccctccatag aagagatgag gaaggtggtg tgtgaccaga ggcttcggcc taatgtgccc
     1081 aactggtggc agagctatga ggcgctgaga gtgatgggaa agatcatgcg ggagtgttgg
     1141 tatgccaacg gagcggcgcg gcttacagct ctgcggatta agaagactct ttcgcaactc
     1201 agcgtccaag aagacattaa gggtggagga ggttctggag gcggtggaag tggtggcgga
     1261 ggtagccctg actacagtct cgtgaaggct ctgcaaatgg cacaacagaa ttttgtcatt
     1321 acagacgcct ccctcccaga caaccctatc gtctacgcca gtagagggtt tctgacactg
     1381 acaggctatt ctctcgacca gatcctgggc aggaactgca ggtttctgca agggccagaa
     1441 acagacccaa gagctgtgga taagatcagg aatgccatca ccaaaggcgt tgataccagt
     1501 gtctgtctgc tgaattatag acaggatggc acaaccttct ggaatctctt cttcgtggct
     1561 ggactcagag attctaaggg caatattgtc aactacgtcg gagtgcagtc aaaggtgagc
     1621 gaagattatg ccaagctgct ggtcaacgag cagaacattg agtacaaagg tgtgcgcacc
     1681 agtaacatgc tgcgcagaaa gcccgggtct agttatccgt acgacgtacc agactacgca
     1741 ggaagcggag ctacaaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct
     1801 ggacctatgg ggagtagcaa gagcaagcct aaggacccca gccagcgcgg tggaggaggt
     1861 tctggaggcg gtggaagtgg tggcggaggt agcaaacctc cgtacggaca tgtggacgtc
     1921 aatgaggatc caggcccatc tcctccatct cctctggtgg gtctgaagcc tctgcagctg
     1981 ctggaggtta aagctcgcgg acgcttcggc tgcgtctgga aggctcagat gatcaatgaa
     2041 tatgtagctg tcaagatttt ccccattcag gataagctgt cgtggcagaa cgagcgggag
     2101 atgttttcca ctccgggaat gaaacatgat aacctgctgc gcttcatcgc tgctgagaaa
     2161 cgcggatcta acctggagat ggagttctgg ctcatcactg agtttcatga gcggggctct
     2221 ctgacggact atctgaaggg gaacgcagtg agctgggctg atctgtgtgt gatagcggag
     2281 agcatggcct gtggtctggc gtatctgcat gaagacgtgc cgcgctccaa aggagaaggc
     2341 cccaaaccag ccatcgcaca cagagacttc aagagcaaga atgtgatgct gaagatggac
     2401 ctcaccgccg tcattgggga ttttgggctg gcggtgcggt ttgagccggg gaaaccgccg
     2461 ggagacacac atggccaggt gggcacgagg aggtacatgg ccccggaggt tctggaagga
     2521 gccataaact tccagcggga ctcctttctg cggatagaca tgtacgccat gggcctggtg
     2581 ctgtgggagc tggtgtcacg ctgcaaagct gctgatggtc ctgtggacga gtacatgctg
     2641 ccgtttgagg aggagatcgg tcagcacccg tcgctggagg atctgcagga tgctgtggtc
     2701 cataagaagc tgcggccggc gtttaaggac tgctggctca agcattcagg tctgtgtcag
     2761 atgtgcgaga ccatggagga gtgctgggat catgacgcag aggctcgtct gtcggccggc
     2821 tgtgtgcagg agcgcatctc tcagatccgc cgcgtcagca gctccacctc agactgcctg
     2881 ttctccatgg tgacctcgct caccaacgtg gacctgccgc ccaaagagtc cagcatcggc
     2941 ggcggcggca gtggcggcgg cggctctggc ggcggcggct ctcctgacta cagtctcgtg
     3001 aaggctctgc aaatggcaca acagaatttt gtcattacag acgcctccct cccagacaac
     3061 cctatcgtct acgccagtag agggtttctg acactgacag gctattctct cgaccagatc
     3121 ctgggcagga actgcaggtt tctgcaaggg ccagaaacag acccaagagc tgtggataag
     3181 atcaggaatg ccatcaccaa aggcgttgat accagtgtct gtctgctgaa ttatagacag
     3241 gatggcacaa ccttctggaa tctcttcttc gtggctggac tcagagattc taagggcaat
     3301 attgtcaact acgtcggagt gcagtcaaag gtgagcgaag attatgccaa gctgctggtc
     3361 aacgagcaga acattgagta caaaggtgtg cgcaccagta acatgctgcg cagaaagccc
     3421 gggtctagtg actacaagga cgacgacgac aagtgatcta gaactatagt gagtcgtatt
     3481 acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     3541 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     3601 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     3661 gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc
     3721 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     3781 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccgggagcat
     3841 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3901 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3961 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4021 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4081 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4141 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4201 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4261 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4321 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4381 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4441 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4501 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4561 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4621 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4681 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4741 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4801 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4861 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4921 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4981 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5041 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5101 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5161 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5221 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5281 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5341 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5401 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5461 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5521 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5581 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5641 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5701 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5761 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5821 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5881 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     5941 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     6001 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     6061 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     6121 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     6181 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     6241 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     6301 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     6361 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     6421 tcggtgcggg cctcttcgct attacgccag tcgaccgcca attcaatatg gcgtatatgg
     6481 actcatgcca attcaatatg gtggatctgg acctgtgcca attcaatatg gcgtatatgg
     6541 actcgtgcca attcaatatg gtggatctgg accccagcca attcaatatg gcggacttgg
     6601 caccatgcca attcaatatg gcggacctgg cactgtgcca actggggagg ggtctacttg
     6661 gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc aagtccgcca tattgaattg
     6721 gcatggtgcc aataatggcg gccatattgg ctatatgcca ggatcaatat ataggcaata
     6781 tccaatatgg ccctatgcca atatggctat tggccaggtt caatactatg tattggccct
     6841 atgccatata gtattccata tatgggtttt cctattgacg tagatagccc ctcccaatgg
     6901 gcggtcccat ataccatata tggggcttcc taataccgcc catagccact cccccattga
     6961 cgtcaatggt ctctatatat ggtctttcct attgacgtca tatgggcggt cctattgacg
     7021 tatatggcgc ctcccccatt gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc
     7081 attgacgtca ataggaccac ccaccattga cgtcaatggg atggctcatt gcccattcat
     7141 atccgttctc acgcccccta ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat
     7201 caatatctat taatagtaac ttggcaagta cattactatt ggaagtacgc cagggtacat
     7261 tggcagtact cccattgacg tcaatggcgg taaatggccc gcgatggctg ccaagtacat
     7321 ccccattgac gtcaatgggg aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg
     7381 ggcggtaggc gtgcctaatg ggaggtctat ataagcaatg ctcgtttagg gaac
//
