LOCUS       sequence-65816-e        6854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rescues the phenotypes caused by LGN RNAi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6854)
  AUTHORS   Kiyomitsu T, Cheeseman IM
  TITLE     Cortical dynein and asymmetric membrane elongation coordinately 
            position the spindle in anaphase.
  JOURNAL   Cell. 2013 Jul 18;154(2):391-402. doi: 10.1016/j.cell.2013.06.010.
  PUBMED    23870127
REFERENCE   2  (bases 1 to 6854)
  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: 
            "10.1016/j.cell.2013.06"; journalName: "Cell"; date: "2013-07-18- 
            18"; volume: "154"; issue: "2"; pages: "391-402"
COMMENT     Sequence Label: pTK47_mCherry-LGN-hardened
FEATURES             Location/Qualifiers
     source          1..6854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             613..1320
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(758..776)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1026..1045
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1366..1386
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1399..1443
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    3642..3763
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3679..3698)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3733..3752
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3770..4225)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3907..3928)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4119..4138
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4252..4356
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4358..4715
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4358..4549
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4381..4401)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4566..4701
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4750..5544
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4750..5544
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4804..4823)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5414..5433
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5732..5751)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5776..5823
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6152..6740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6641..6660
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
      661 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
      721 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
      781 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
      841 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
      901 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
      961 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     1021 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     1081 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     1141 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     1201 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     1261 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     1321 tccggagggg atcggtggag ctccaccggt ggcggccgct ctagagagaa tctttatttt
     1381 cagggcgccg ccaaattcaa agaaaccgct gctgctaaat tcgaacgcca gcacatggac
     1441 agcggaggtg gaggtactag tctcgagatg agagaagacc attcttttca tgttcgttac
     1501 agaatggaag cttcttgcct agagctggcc ttggaagggg aacgtctatg taaatcagga
     1561 gactgccgcg ctggcgtgtc attctttgaa gctgcagttc aagttggaac tgaagaccta
     1621 aaaacactta gcgctattta cagccagttg ggcaatgctt atttctattt gcatgattat
     1681 gccaaagcat tagaatatca ccatcatgat ttaacccttg caaggactat tggagaccag
     1741 ctgggggaag cgaaagctag tggtaatctg ggaaacacct taaaagttct tgggaatttt
     1801 gacgaagcca tagtttgttg tcagcgacac ctagatattt ccagagagct taatgacaag
     1861 gtgggagaag caagagcact ttacaatctt gggaatgtgt atcatgccaa agggaaaagt
     1921 tttggttgcc ctggtcccca ggatgtagga gaatttccag aagaagtgag agatgctctg
     1981 caggcagccg tggattttta tgaggaaaac ctatcattag tgactgcttt gggtgaccga
     2041 gcggcacaag gacgtgcctt tggaaatctt ggaaacacac attacctcct tggcaacttc
     2101 agggatgcag ttatagctca tgagcagcgt ctccttattg caaaagaatt tggagataaa
     2161 gcagctgaaa gaagagcata tagcaacctt ggaaatgcat atatatttct tggtgaattt
     2221 gaaactgcct cggaatacta caagaagaca ctactgttgg cccgacagct taaagaccga
     2281 gctgtagaag cacagtcttg ttacagtctt ggaaatacat atactttact tcaagactat
     2341 gaaaaggcca ttgattatca tctgaagcac ttagcaattg ctcaagagct gaatgataga
     2401 attggtgaag gaagagcatg ttggagctta ggaaatgcat acacagcact aggaaatcat
     2461 gatcaagcaa tgcattttgc tgaaaagcac ttggaaattt caagagaggt tggggataaa
     2521 agtggtgagt tgactgctcg tttaaatctc tcagaccttc aaatggttct tggtctgagc
     2581 tacagcacaa ataactccat aatgtctgaa aatactgaaa ttgatagcag tttgaatggt
     2641 gtacgcccca agttgggacg ccggcatagt atggaaaata tggaacttat gaagttaaca
     2701 ccagaaaagg tacagaactg gaacagtgaa attcttgcta agcaaaaacc tcttattgcc
     2761 aaaccttctg caaagctact ctttgtcaac agactgaagg ggaaaaaata caaaacgaat
     2821 tcctccacta aagttctcca agatgccagt aattctattg accaccgaat tccaaattct
     2881 cagaggaaaa tcagtgcaga tactattgga gatgaagggt tctttgactt attaagccga
     2941 tttcaaagca ataggatgga tgatcagaga tgttgcttac aagaaaagaa ctgccataca
     3001 gcttcaacaa caacttcttc cactccccct aaaatgatgc taaaaacatc atctgttcct
     3061 gtggtatccc ccaacacgga tgagttttta gatcttcttg ccagctcaca gagtcgccgt
     3121 ctggatgacc agagggctag tttcagtaat ttgccagggc ttcgtctaac acaaaacagc
     3181 cagtcggtac ttagccacct gatgactaat gacaacaaag aggctgatga agatttcttt
     3241 gacatccttg taaaatgtca aggatccaga ttagatgatc aaagatgtgc tccaccacct
     3301 gctaccacaa agggtccgac agtaccagat gaagactttt tcagccttat tttacggtcc
     3361 cagggaaaga gaatggatga acagagagtt cttttacaaa gagatcaaaa cagagacact
     3421 gactttgggc taaaggactt tttgcaaaat aatgctttgt tggagtttaa aaattcaggg
     3481 aaaaaatcgg cagaccatta gccgcgggcc cgggatccac cggatctaga taactgatca
     3541 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     3601 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     3661 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3721 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc
     3781 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3841 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3901 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3961 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     4021 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     4081 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     4141 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     4201 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4261 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4321 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     4381 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4441 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     4501 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4561 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4621 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4681 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     4741 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4801 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4861 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4921 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4981 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     5041 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     5101 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     5161 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     5221 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     5281 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     5341 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     5401 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     5461 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     5521 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5581 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5641 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5701 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     5761 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     5821 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     5881 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     5941 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     6001 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6061 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6121 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6181 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6241 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     6301 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6361 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6421 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6481 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6541 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6601 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6661 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6721 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6781 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6841 ttaccgccat gcat
//
