LOCUS       sequence-176859-        6904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express NES-PhoCl-mCherry in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6904)
  AUTHORS   Zhang W, Lohman AW, Zhuravlova Y, Lu X, Wiens MD, Hoi H, Yaganoglu 
            S, Mohr MA, Kitova EN, Klassen JS, Pantazis P, Thompson RJ, Campbell
            RE
  TITLE     Optogenetic control with a photocleavable protein, PhoCl.
  JOURNAL   Nat Methods. 2017 Mar 13. doi: 10.1038/nmeth.4222.
  PUBMED    28288123
REFERENCE   2  (bases 1 to 6904)
  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: "Nat 
            Methods. 2017 Mar 13. doi: 10.1038/nmeth.4222."
COMMENT     Sequence Label: pcDNA-NES-PhoCl-mCherry
FEATURES             Location/Qualifiers
     source          1..6904
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        495..698
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     649..669
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     743..762
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        743..761
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             859..1584
                     /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"
     CDS             1630..2337
                     /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(1772..1790)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2040..2059
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2375..2392)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2381..2605
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2651..3079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2738..2757)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2948..2969
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3088..3108)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3093..3422
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3273..3408
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3335..3354
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3489..4283
                     /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(3543..3562)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4153..4172
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4457..4578
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4494..4513)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4548..4567
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4627..4643)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4627..4643)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4640..4662)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4651..4667
                     /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(4675..4681,4682..4699,4700..4705))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4720..4741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4858..4875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5029..5617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5109..5128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5788..6579,6580..6648))
                     /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     6411..6430
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6649..6753)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6828..6847)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
       61 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      121 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      181 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      241 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      301 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      361 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      421 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      481 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      541 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      601 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      661 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      721 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      781 gccaccatga acctggtgga cctgcagaag aagctggagg agctggagct ggacgagcag
      841 cagggaggcg ggggcagcgt gatccctgac tacttcaagc agagcttccc cgagggctac
      901 agctgggagc gcagcatgac ctacgaggac ggcggcatct gcatcgccac caacgacatc
      961 acaatggagg gggacagctt catcaacaag atccacttca agggcacgaa cttccccccc
     1021 aacggccccg tgatgcagaa gaggaccgtg ggctgggagg ccagcaccga gaagatgtac
     1081 gagcgcgacg gcgtgctgaa gggcgacgtg aagatgaagc tgctgctgaa gggcggcggc
     1141 cactatcgct gcgactaccg caccacctac aaggtcaagc agaagcccgt aaagctgccc
     1201 gactaccact tcgtggacca ccgcatcgag atcctgagcc acgacaagga ctacaacaag
     1261 gtgaagctgt acgagcacgc cgtggcccgc aactccaccg acagcatgga cgagctgtac
     1321 aagggtggca gcggtggcat ggtgagcaag ggcgaggaga ccattacaag cgtgatcaag
     1381 cctgacatga agaacaagct gcgcatggag ggcaacgtga acggccacgc cttcgtgatc
     1441 gagggcgagg gcagcggcaa gcccttcgag ggcatccaga cgattgattt ggaggtgaag
     1501 gagggcgccc cgctgccctt cgcctacgac atcctgacca ccgccttcca ctacggcaac
     1561 cgcgtgttca ccaagtaccc acggggaggt ggaggtaccg gaggcggggg cagcggaggc
     1621 gggggcagcg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     1681 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     1741 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     1801 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     1861 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     1921 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     1981 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     2041 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     2101 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     2161 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     2221 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     2281 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaactc
     2341 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     2401 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2461 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2521 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2581 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2641 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2701 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2761 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2821 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2881 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2941 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3001 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3061 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3121 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3181 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3241 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3301 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3361 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3421 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     3481 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3541 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     3601 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     3661 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     3721 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     3781 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     3841 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     3901 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     3961 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     4021 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4081 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4141 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4201 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4261 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4321 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4381 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4441 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4501 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4561 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4621 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4681 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4741 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4801 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4861 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4921 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4981 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5041 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5101 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5161 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5221 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5281 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5341 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5401 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5461 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5521 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5581 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5641 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5701 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5761 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5821 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5881 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5941 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6001 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6061 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6121 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6181 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6241 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6301 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6361 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6421 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6481 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6541 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6601 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6661 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6721 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6781 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     6841 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     6901 tgcg
//
