LOCUS       sequence-269826-        7661 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 7661)
  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 7661)
  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-synPCB2.2
FEATURES             Location/Qualifiers
     source          1..7661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..665
                     /label=chicken beta-actin promoter
     intron          666..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             1733..1807
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             3038..3094
                     /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             3095..3169
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      4360..4369
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4649..4672
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4685..4704)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4750..4805
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4804..4823)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5164..5180)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5164..5180)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5177..5199)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5188..5204
                     /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(5212..5218,5219..5236,5237..5242))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5257..5278
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5337..5532
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5383..5518
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5538..5672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5575..5594)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5629..5648
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5740..5757)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5911..6499)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5991..6010)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6670..7461,7462..7530))
                     /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     7293..7312
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7531..7635)
                     /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 gactatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cgggggcgag
      541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
      841 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtgcggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctggcgg agccgaaatc tgggaggcgc cgccgcaccc
     1441 cctctagcgg gcgcgggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg
     1501 ccttcgtgcg tcgccgcgcc gccgtcccct tctccatctc cagcctcggg gctgccgcag
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggttg ttgtgctgtc tcatcatttt ggcaagaatt caatgtccgt
     1741 cctgacgccg ctgctgctgc ggggcttgac aggctcggcc cggcggctcc cagtgccgcg
     1801 cgccaagatc cattcgttgc tcgagaatat ttaccgacct aacaatcctt gcattgggaa
     1861 agtcattagc aatgaggagc tggtgcgaga ggggggagaa gggactgtaa aacatataat
     1921 cttcgatata tcaggcacgg agcttcggta tcttgagggg caatctattg gaatcatacc
     1981 ggcgggtact gatgccaatg gaaaaccgca taaattgcgc ctctactcta tagcttctac
     2041 taggcacggg gatttccagg atgataagac cgtctctttg tgtgtccgca ggctggaata
     2101 caaggataag gaaaccggag agacaatata tggggtatgt tcctcatacc tgaatcaatt
     2161 gcaaccagga gatgaagtga agataacggg gcctgttggt aaggagatgc tcctgtcaga
     2221 tgacccagaa gctacaatta taatgctggc gactgggact ggcatagcgc ctttccgagc
     2281 cttcctctgg agaatgttca aggaaaacaa ccccgattat caattcaagg ggcttgcctg
     2341 gttgtttttt ggtgtagcat acacggctaa catcctttac aaagatgaac tcgaagcgat
     2401 tcaggcgcag tacccggatc attttcgctt gacatacgcc atatctaggg aacagaagac
     2461 ccccgacggg ggtaaaatgt atatccaagg gcgaatagcc gaacatgcag atgaaatctg
     2521 gcaactgctg caaaagaaaa atactcacgt ctatatgtgt ggtctcagag gaatggagcc
     2581 gggcatagac gaagcgatga ccgctgcggc ggcaaagaac ggcgccgatt ggcaagaatt
     2641 tctcaagggt acacttaaga aggaggggcg ctggcacgtc gagacgtacg gcggatccgg
     2701 aggttccggc ggctcaggag gttctatggc tacctataag gtcacgctgg ttcgaccaga
     2761 tggtagcgag actaccattg atgtccctga agacgaatat attctggacg tagctgagga
     2821 gcaaggcttg gatcttcctt tcagctgccg ggctggtgct tgcagcacgt gcgcgggaaa
     2881 acttctggag ggagaggtcg atcagagcga tcagtccttc ctcgacgatg accaaataga
     2941 aaagggtttc gttctcactt gcgttgccta cccaagaagc gattgcaaaa tactcacaaa
     3001 ccaggaggaa gagctgtacg gcggccgcgg ctcaggtgcg acgaatttta gtctactgaa
     3061 acaagcggga gacgtggagg aaaaccctgg acctatgtca gtgctgactc cactgctgct
     3121 gaggggcctg actgggtccg cacgccgcct gcctgtccca agagccaaaa tccatagcct
     3181 gggcgggtct ggaggcatga ctaccagcct ggctacaaag ctgagggaag gcactaagaa
     3241 agctcatacc atggcagaga acgtggggtt tgtccggtgt ttcctgaagg gaactgtgga
     3301 gaaaagctcc tatagaaagc tggtggcctc tctgtaccac gtctatagtg ctatggagca
     3361 ggaaatggag cgcctgaaag accatccaat cgtgggcaaa atctactttc ccgaactgaa
     3421 caggaagtct agtctggagc gcgatctgac atactatttc gggtccaatt ggagggagga
     3481 aattccccct tctcccgcaa ctcaggccta cgtggctcgc atccacgaag tcgcaaacac
     3541 cgcccctgag ctgctggtgg cccatagtta cacacggtat ctgggagacc tgtcaggggg
     3601 acagatcctg aagggcattg ctgaacgggc aatgaatctg caggatgggg agggaaccgc
     3661 cttctacaga ttcgaaagta tctcagacga gaaggccttc aaacagctgt atcgacagcg
     3721 gctggatgaa ctgcccgtgg acgaggctac agcagataga attgtggacg aggctaacgc
     3781 tgcatttggc atgaatatga agattttcca ggaactggag ggcaacctga tccgggccat
     3841 tgggcagctg ctgtttaata ccctgacacg acggaaacag agaggcagca ccgagctggc
     3901 cacagctgat ggtggaggtg gatctggagg tggtggatca atgagtctga ggcagcacca
     3961 gcatcctctg atccagcgcc tggccgatcg aatcgaggca atttggcagg ccttctttcc
     4021 cctggcacct tacgccctgc cagaagacct gggctatgtg gagggaaaac tggagggcga
     4081 acgcctgaca atcgagaacc actgctatca ggccccccct tttcggaagc tgcatctgga
     4141 actggctaga gtgggcgagt ctctggacat tctgcactgc gtcatgtttc ccgagcctag
     4201 atacgatctg cccatgttcg gatgtgacct ggtgggggga cgcggacaga tcagtgccgc
     4261 tattgtggat ctgtcaccag tcactggaca gctgccagca gcatacacct gcgctctgaa
     4321 tgcactgcct aagctgacct ttagacagcc aagggagctg ccaccatggg gacatatctt
     4381 ctctcccttt tgtatcttca ttagacctca gggcgaagcc gaggaacagc agttcctgga
     4441 taggattggg gagtatctga ctctgcactg ccagctgagc cagcaggcag tgcctaccga
     4501 ccatccacag gccgtcatcg ctggacagcg gcagtattgt cagcagcagc agcagaacga
     4561 taaaactcgg agagtgctgg aaaaggcttt tggcgtccca tgggcagaga gatacatgac
     4621 cacagtgctg ttcgatgtgc ctccagtgga ctataaagac gatgacgata aataagtcga
     4681 cggatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga
     4741 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc
     4801 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg
     4861 tttagagttt ggcaacatat gccatatgct ggctgccatg aacaaaggtg gctataaaga
     4921 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg
     4981 acttgaggtt agattttttt ttatattttg tttgtgttat ttttttcttt aacatcccta
     5041 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc
     5101 atagctgtcc ctcttctctt atgaagatcc ctcgacctgc agcccaagct tggcgtaatc
     5161 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5221 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5281 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat
     5341 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     5401 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     5461 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     5521 ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5581 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5641 aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc
     5701 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5761 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5821 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5881 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5941 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6001 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6061 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     6121 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6181 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6241 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6301 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     6361 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6421 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6481 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6541 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6601 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6661 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6721 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6781 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6841 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6901 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6961 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7021 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7081 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7141 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7201 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7261 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7321 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7381 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7441 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7501 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7561 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7621 aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
