LOCUS       sequence-322532-        6895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Optogenetic control of protein localization by PhoCl2f.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6895)
  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 6895)
  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-NES-PhoCl2f-mCherry
FEATURES             Location/Qualifiers
     source          1..6895
                     /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"
     CDS             979..1704
                     /codon_start=1
                     /product="photocleavable protein that separates into two 
                     fragments upon exposure to violet light (Zhang et al., 
                     2017)"
                     /label=PhoCl
                     /translation="VIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKI
                     HFQGTNFPPNGPVMQKRTVGWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRCDYRTTY
                     KVKQKPVKLPDCHFVDHRIEILSHDKDYNKVKLYEHAVAKTSTDSMDELYKGGSGGMVS
                     KGEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGSQTIDLEVKEGAPLPFA
                     YDILTTAFHYGNRVFTKYPR"
     CDS             1741..2448
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1883..1901)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2151..2170
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2486..2503)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2492..2716
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2762..3190
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2849..2868)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3059..3080
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3199..3219)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3204..3533
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3384..3519
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3446..3465
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3600..4394
                     /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(3654..3673)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4264..4283
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4568..4689
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4605..4624)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4659..4678
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4738..4754)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4738..4754)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4751..4773)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4762..4778
                     /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(4786..4792,4793..4810,4811..4816))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4831..4852
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4969..4986)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5140..5728)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5220..5239)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5899..6690,6691..6759))
                     /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     6522..6541
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6760..6864)
                     /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 gccaccatga acctggtgga cctgcagaag aagctggagg agctggagct ggacgagcag
      961 cagggaggca agcttagcgt gatccctgac tacttcaagc agagcttccc cgagggctac
     1021 agctgggagc gcagcatgac ctacgaggac ggcggcatct gcatcgccac caacgacatc
     1081 acaatggagg gggacagctt catcaacaag atccacttcc agggcacgaa cttccccccc
     1141 aacggccccg tgatgcagaa gaggaccgtg ggctgggagg ccagcaccga gaagatgtac
     1201 gagcgcgacg gcgtgctgaa gggcgacgtg aagatgaagc tgctgctgaa gggcggcggc
     1261 cactatcgct gcgactaccg caccacctac aaggtcaagc agaagcccgt aaagctgccc
     1321 gactgccact tcgtggacca ccgcatcgag atcctgagcc acgacaagga ctacaacaag
     1381 gtgaagctgt acgagcacgc cgtggccaag acttccaccg acagcatgga cgagctgtac
     1441 aagggtggca gcggtggcat ggtgagcaag ggcgaggaga ccattacaag cgtgatcaag
     1501 cctgacatga agaacaagct gcgcatggag ggcaacgtga acggccacgc cttcgtgatc
     1561 gagggcgagg gcagcggcaa gcccttcgag ggctctcaga cgattgattt ggaggtgaag
     1621 gagggcgccc cgctgccctt cgcctacgac atcctgacca ccgccttcca ctacggcaac
     1681 cgcgtgttca ccaagtaccc acggggtacc ggaggcgggg gcagcggagg cgggggcagc
     1741 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     1801 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1861 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1921 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     1981 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     2041 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     2101 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     2161 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     2221 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     2281 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     2341 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     2401 gccgagggcc gccactccac cggtggcatg gacgagctgt acaagtaact cgagtctaga
     2461 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2521 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2581 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2641 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2701 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2761 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2821 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2881 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2941 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3001 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3061 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3121 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3181 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3241 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3301 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3361 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3421 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3481 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3541 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     3601 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3661 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3721 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3781 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3841 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3901 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     3961 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4021 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4081 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     4141 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4201 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4261 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4321 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4381 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4441 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4501 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4561 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4621 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4681 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     4741 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4801 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4861 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4921 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4981 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5041 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5101 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5161 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5221 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5281 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5341 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5401 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5461 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5521 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5581 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5641 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5701 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5761 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5821 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5881 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5941 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6001 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6061 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6121 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6181 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6241 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6301 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6361 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6421 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6481 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6541 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6601 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6661 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6721 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6781 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     6841 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
