LOCUS       sequence-275166-        7901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for phycocyanobilin (PCB) synthesis in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7901)
  AUTHORS   Uda Y, Miura H, Goto Y, Yamamoto K, Mii Y, Kondo Y, Takada S, Aoki K
  TITLE     Improvement of Phycocyanobilin Synthesis for Genetically Encoded 
            Phytochrome-Based Optogenetics.
  JOURNAL   ACS Chem Biol. 2020 Nov 20;15(11):2896-2906. doi: 
            10.1021/acschembio.0c00477. Epub 2020 Nov 9.
  PUBMED    33164485
REFERENCE   2  (bases 1 to 7901)
  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: 
            "10.1021/acschembio.0c00477"; journalName: "ACS Chem Biol"; date: 
            "2020-11-20- 20"; volume: "15"; issue: "11"; pages: "2896-2906"
COMMENT     Sequence Label: pCAGGS-synPCB2.0
FEATURES             Location/Qualifiers
     source          1..7901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        389..664
                     /label=chicken beta-actin promoter
     intron          665..1685
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1605..1627
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1693..1712
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1739..1813
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      2260..2269
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2549..2572
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2582..2638
                     /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             2642..2716
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2747..2773
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3509..3565
                     /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             3566..3640
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     primer_bind     complement(4922..4941)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4987..5042
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5041..5060)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5403..5419)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5403..5419)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5416..5438)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5427..5443
                     /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(5451..5457,5458..5475,5476..5481))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5496..5517
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5576..5771
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5622..5757
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5777..5911
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5814..5833)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5868..5887
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5979..5996)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6150..6738)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6230..6249)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6909..7700,7701..7769))
                     /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     7532..7551
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7770..7874)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgatcgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
      421 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
      481 gcagcgatgg gggcgggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg
      541 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      901 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      961 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     1021 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1081 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1141 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1201 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1261 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc
     1321 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1381 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1441 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1501 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1561 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1621 tgtgaccggc ggctctagct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
     1681 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aagaattcat
     1741 gtcagtgctg actccactgc tgctgagggg cctgactggg tccgcacgcc gcctgcctgt
     1801 cccaagagcc aaaatccata gcctgggcgg gtctggaggc atgagtctga ggcagcacca
     1861 gcatcctctg atccagcgcc tggccgatcg aatcgaggca atttggcagg ccttctttcc
     1921 cctggcacct tacgccctgc cagaagacct gggctatgtg gagggaaaac tggagggcga
     1981 acgcctgaca atcgagaacc actgctatca ggccccccct tttcggaagc tgcatctgga
     2041 actggctaga gtgggcgagt ctctggacat tctgcactgc gtcatgtttc ccgagcctag
     2101 atacgatctg cccatgttcg gatgtgacct ggtgggggga cgcggacaga tcagtgccgc
     2161 tattgtggat ctgtcaccag tcactggaca gctgccagca gcatacacct gcgctctgaa
     2221 tgcactgcct aagctgacct ttagacagcc aagggagctg ccaccatggg gacatatctt
     2281 ctctcccttt tgtatcttca ttagacctca gggcgaagcc gaggaacagc agttcctgga
     2341 taggattggg gagtatctga ctctgcactg ccagctgagc cagcaggcag tgcctaccga
     2401 ccatccacag gccgtcatcg ctggacagcg gcagtattgt cagcagcagc agcagaacga
     2461 taaaactcgg agagtgctgg aaaaggcttt tggcgtccca tgggcagaga gatacatgac
     2521 cacagtgctg ttcgatgtgc ctccagtgga ctataaagac gatgacgata aaggcagcgg
     2581 agcgacgaat tttagtctac taaaacaagc gggtgatgta gaagaaaatc cgggtccggg
     2641 catgtcagtt ctgacacctc tgctgctgcg tggactgact ggaagcgcca ggcgcctgcc
     2701 tgtgccacga gcaaaaatcc actcactggg cgggagcgga ggcatgtacc cctatgatgt
     2761 ccctgactac gcctctagaa tgactaccag cctggctaca aagctgaggg aaggcactaa
     2821 gaaagctcat accatggcag agaacgtggg gtttgtccgg tgtttcctga agggaactgt
     2881 ggagaaaagc tcctatagaa agctggtggc ctctctgtac cacgtctata gtgctatgga
     2941 gcaggaaatg gagcgcctga aagaccatcc aatcttgggc aaaatctact ttcccgaact
     3001 gaacaggaag tctagtctgg agcgcgatct gacatactat ttcgggtcca attggaggga
     3061 ggaaattccc ccttctcccg caactcaggc ctacgtggct cgcatccacg aagtcgcaaa
     3121 caccgcccct gagctgctgg tggcccatag ttacacacgg tatctgggag acctgtcagg
     3181 gggacagatc ctgaagggca ttgctgaacg ggcaatgaat ctgcaggatg gggagggaac
     3241 cgccttctac agattcgaaa gtatctcaga cgagaaggcc ttcaaacagc tgtatcgaca
     3301 gcggctggat gaactgcccg tggacgaggc tacagcagat agaattgtgg acgaggctaa
     3361 cgctgcattt ggcatgaata tgaagatttt ccaggaactg gagggcaacc tgatccgggc
     3421 cattgggcag ctgctgttta ataccctgac acgacggaaa cagagaggca gcaccgagct
     3481 ggccacagct gatagatctg gctcaggtgc gacgaatttt agtctactga aacaagcggg
     3541 agacgtggag gaaaaccctg gacctatgtc cgtcctgacc ccactgctgc tgaggggact
     3601 gacaggatct gcaagaaggc tgccagtgcc cagagccaaa attcactccc tgggaggctc
     3661 tggaggcctc gagaatattt accgacctaa caatccttgc attgggaaag tcattagcaa
     3721 tgaggagctg gtgcgagagg ggggagaagg gactgtaaaa catataatct tcgatatatc
     3781 aggcacggag cttcggtatc ttgaggggca atctattgga atcataccgg cgggtactga
     3841 tgccaatgga aaaccgcata aattgcgcct ctactctata gcttctacta ggcacgggga
     3901 tttccaggat gataagaccg tctctttgtg tgtccgcagg ctggaataca aggataagga
     3961 aaccggagag acaatatatg gggtatgttc ctcatacctg aatcaattgc aaccaggaga
     4021 tgaagtgaag ataacggggc ctgttggtaa ggagatgctc ctgtcagatg acccagaagc
     4081 tacaattata atgctggcga ctgggactgg catagcgcct ttccgagcct tcctctggag
     4141 aatgttcaag gaaaacaacc ccgattatca attcaagggg cttgcctggt tgttttttgg
     4201 tgtagcatac acggctaaca tcctttacaa agatgaactc gaagcgattc aggcgcagta
     4261 cccggatcat tttcgcttga catacgccat atctagggaa cagaagaccc ccgacggggg
     4321 taaaatgtat atccaagggc gaatagccga acatgcagat gaaatctggc aactgctgca
     4381 aaagaaaaat actcacgtct atatgtgtgg tctcagagga atggagccgg gcatagacga
     4441 agcgatgacc gctgcggcgg caaagaacgg cgccgattgg caagaatttc tcaagggtac
     4501 acttaagaag gaggggcgct ggcacgtcga gacgtacggc ggatccggag gttccggcgg
     4561 ctcaggaggt tctatggcta cctataaggt cacgctggtt cgaccagatg gtagcgagac
     4621 taccattgat gtccctgaag acgaatatat tctggacgta gctgaggagc aaggcttgga
     4681 tcttcctttc agctgccggg ctggtgcttg cagcacgtgc gcgggaaaac ttctggaggg
     4741 agaggtcgat cagagcgatc agtccttcct cgacgatgac caaatagaaa agggtttcgt
     4801 tctcacttgc gttgcctacc caagaagcga ttgcaaaata ctcacaaacc aggaggaaga
     4861 gctgtacggc ggccgcatgg cttcaatgac aggaggacag cagatggggt aagtcgacgg
     4921 atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt
     4981 ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct
     5041 cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt
     5101 agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag
     5161 gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga
     5221 cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa
     5281 aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca
     5341 tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt ggcgtaatca
     5401 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5461 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5521 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc
     5581 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     5641 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     5701 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     5761 gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5821 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5881 actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg
     5941 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     6001 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6061 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6121 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6181 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6241 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6301 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6361 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6421 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6481 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6541 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6601 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6661 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6721 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6781 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6841 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6901 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6961 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     7021 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     7081 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7141 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7201 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7261 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7321 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7381 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7441 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7501 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7561 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7621 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7681 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7741 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7801 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7861 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg g
//
