LOCUS       Exported                6874 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Express NLS-PhoCl-mCherry in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA-NLS-PhoCl-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6874)
  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 6874)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6874
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        150..529
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        530..733
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     684..704
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     778..797
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        778..796
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      816..825
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             828..848
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             864..1589
                     /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             1635..2342
                     /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(1777..1795)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2045..2064
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2380..2397)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2386..2610
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2656..3084
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2743..2762)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2953..2974
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3093..3113)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3098..3427
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3278..3413
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3340..3359
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3494..4288
                     /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(3548..3567)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4158..4177
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4462..4583
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4499..4518)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4553..4572
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4632..4648)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4632..4648)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4645..4667)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4656..4672
                     /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(4680..4710)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4725..4746
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4863..4880)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5034..5622)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5114..5133)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5793..6653)
                     /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     6416..6435
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6654..6758)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6833..6852)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
       61 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
      121 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      181 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      241 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      301 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      361 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      421 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      481 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      541 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      601 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      661 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      721 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
      781 tacgactcac tatagggaga cccaagctgg ctagcgccac catgggccca aaaaagaaaa
      841 gaaaagttgg aggcgggggc agcgtgatcc ctgactactt caagcagagc ttccccgagg
      901 gctacagctg ggagcgcagc atgacctacg aggacggcgg catctgcatc gccaccaacg
      961 acatcacaat ggagggggac agcttcatca acaagatcca cttcaagggc acgaacttcc
     1021 cccccaacgg ccccgtgatg cagaagagga ccgtgggctg ggaggccagc accgagaaga
     1081 tgtacgagcg cgacggcgtg ctgaagggcg acgtgaagat gaagctgctg ctgaagggcg
     1141 gcggccacta tcgctgcgac taccgcacca cctacaaggt caagcagaag cccgtaaagc
     1201 tgcccgacta ccacttcgtg gaccaccgca tcgagatcct gagccacgac aaggactaca
     1261 acaaggtgaa gctgtacgag cacgccgtgg cccgcaactc caccgacagc atggacgagc
     1321 tgtacaaggg tggcagcggt ggcatggtga gcaagggcga ggagaccatt acaagcgtga
     1381 tcaagcctga catgaagaac aagctgcgca tggagggcaa cgtgaacggc cacgccttcg
     1441 tgatcgaggg cgagggcagc ggcaagccct tcgagggcat ccagacgatt gatttggagg
     1501 tgaaggaggg cgccccgctg cccttcgcct acgacatcct gaccaccgcc ttccactacg
     1561 gcaaccgcgt gttcaccaag tacccacggg gaggtggagg taccggaggc gggggcagcg
     1621 gaggcggggg cagcgtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
     1681 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     1741 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     1801 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     1861 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     1921 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     1981 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     2041 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     2101 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     2161 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     2221 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     2281 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     2341 aactcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2401 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2461 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2521 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2581 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2641 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2701 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2761 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2821 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2881 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2941 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3001 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3061 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     3121 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3181 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3241 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3301 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3361 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3421 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     3481 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3541 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3601 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3661 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3721 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     3781 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     3841 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     3901 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     3961 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4021 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4081 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4141 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4201 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4261 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4321 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4381 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4441 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4501 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4561 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4621 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4681 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4741 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4801 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4861 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4921 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4981 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5041 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5101 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5161 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5221 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5281 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5341 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5401 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5461 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5521 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5581 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5641 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5701 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5761 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5821 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5881 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5941 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6001 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6061 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6121 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6181 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6241 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6301 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6361 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6421 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6481 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6541 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6601 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6661 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6721 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6781 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     6841 gctctgatgc cgcatagtta agccagtatc tgct
//
