LOCUS       sequence-322535-        6730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Optogenetic manipulation of cell apoptosis using PhoCl1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6730)
  AUTHORS   Lu X, Wen Y, Zhang S, Zhang W, Chen Y, Shen Y, Lemieux MJ, Campbell 
            RE
  TITLE     Photocleavable proteins that undergo fast and efficient 
            dissociation.
  JOURNAL   Chem Sci. 2021 May 31;12(28):9658-9672. doi: 10.1039/d1sc01059j. 
            eCollection 2021 Jul 21.
  PUBMED    34349937
REFERENCE   2  (bases 1 to 6730)
  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: 
            "10.1039/d1sc01059j"; journalName: "Chem Sci"; date: "2021-05-31- 
            31"; volume: "12"; issue: "28"; pages: "9658-9672"
COMMENT     Sequence Label: pcDNA-NBid-PhoCl1-CBid
FEATURES             Location/Qualifiers
     source          1..6730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1081..1806
                     /codon_start=1
                     /product="photocleavable protein that separates into two 
                     fragments upon exposure to violet light (Zhang et al., 
                     2017)"
                     /label=PhoCl
                     /translation="VIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKI
                     HFKGTNFPPNGPVMQKRTVGWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRCDYRTTY
                     KVKQKPVKLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVS
                     KGEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFA
                     YDILTTAFHYGNRVFTKYPR"
     primer_bind     complement(2321..2338)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2327..2551
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2597..3025
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2684..2703)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2894..2915
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3034..3054)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3039..3368
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3219..3354
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3281..3300
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3435..4229
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3489..3508)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4099..4118
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4403..4524
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4440..4459)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4494..4513
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4573..4589)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4573..4589)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4586..4608)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4597..4613
                     /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(4621..4627,4628..4645,4646..4651))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4666..4687
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4804..4821)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4975..5563)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5055..5074)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5734..6525,6526..6594))
                     /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     6357..6376
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6595..6699)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccaccatgg attgtgaggt caataacggt tcatctctcc gagacgaatg cataacgaac
      961 ttgctcgttt tcggctttct gcaatcctgc agcgataatt ctttcagaag agaacttgac
     1021 gctcttggac atgaactccc agtactcgct ccacagtggg aaggctacga tgagggatcc
     1081 gtgatccctg actacttcaa gcagagcttc cccgagggct acagctggga gcgcagcatg
     1141 acctacgagg acggcggcat ctgcatcgcc accaacgaca tcacaatgga gggggacagc
     1201 ttcatcaaca agatccactt caagggcacg aacttccccc ccaacggccc cgtgatgcag
     1261 aagaggaccg tgggctggga ggccagcacc gagaagatgt acgagcgcga cggcgtgctg
     1321 aagggcgacg tgaagatgaa gctgctgctg aagggcggcg gccactatcg ctgcgactac
     1381 cgcaccacct acaaggtcaa gcagaagccc gtaaagctgc ccgactacca cttcgtggac
     1441 caccgcatcg agatcctgag ccacgacaag gactacaaca aggtgaagct gtacgagcac
     1501 gccgtggccc gcaactccac cgacagcatg gacgagctgt acaagggtgg cagcggtggc
     1561 atggtgagca agggcgagga gaccattaca agcgtgatca agcctgacat gaagaacaag
     1621 ctgcgcatgg agggcaacgt gaacggccac gccttcgtga tcgagggcga gggcagcggc
     1681 aagcccttcg agggcatcca gacgattgat ttggaggtga aggagggcgc cccgctgccc
     1741 ttcgcctacg acatcctgac caccgccttc cactacggca accgcgtgtt caccaagtac
     1801 ccacggggta cccttcaaac cgatggaaac cgatcatctc attcaaggtt ggggcgaatt
     1861 gaagcagata gtgagagtca ggaggacata atacgcaata tagctcgaca ccttgcacag
     1921 gtcggtgata gcatggaccg ctctatcccc ccaggtttgg ttaacggatt ggccctgcaa
     1981 ctgcgaaaca cttcaaggag tgaagaagat agaaatcgag acctggcgac cgcgcttgaa
     2041 cagcttcttc aagcctatcc cagagatatg gagaaggaaa aaacaatgct cgtgctcgca
     2101 ctgctgctgg caaagaaagt agcctctaac acaccatccc tcttgagaga cgtcttccat
     2161 accacggtaa atttcataaa ccagaacctt aggacgtatg tgcggagtct tgctcgcaat
     2221 ggtatgactg agtttcttca acccgtgccc ctcaatgcgt ggttgctcag gttgaacaag
     2281 taactcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     2341 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2401 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2461 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2521 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2581 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2641 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2701 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2761 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2821 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2881 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2941 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3001 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     3061 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3121 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3181 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3241 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3301 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3361 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     3421 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3481 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3541 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3601 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3661 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3721 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3781 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3841 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3901 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3961 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4021 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4081 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4141 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4201 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     4261 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     4321 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4381 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     4441 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4501 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     4561 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4621 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4681 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4741 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     4801 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4861 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4921 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4981 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5041 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5101 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5161 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5221 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5281 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5341 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5401 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5461 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5521 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5581 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5641 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5701 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5761 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5821 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5881 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5941 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6001 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6061 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6121 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6181 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6241 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6301 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6361 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6421 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6481 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6541 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6601 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6661 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6721 acctgacgtc
//
