LOCUS       sequence-430215-        7323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual cistronic expression vector for the RAPID-release approach. 
            H3.2-mCherry is released from mitochondria to the nucleus upon 
            addition of rapamycin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7323)
  AUTHORS   Pardal AJ, Bowman AJ
  TITLE     A specific role for importin-5 and NASP in the import and nuclear 
            hand-off of monomeric H3.
  JOURNAL   Elife. 2022 Sep 6;11:e81755. doi: 10.7554/eLife.81755.
  PUBMED    36066346
REFERENCE   2  (bases 1 to 7323)
  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: "Elife."; 
            date: "2022-09-6"; volume: "11:e81755. doi"; pages: " 
            10.7554/eLife.81755"
COMMENT     Sequence Label: pCL1177
FEATURES             Location/Qualifiers
     source          1..7323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     144..163
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(462..481)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    506..553
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      882..1470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1371..1390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1645..1948
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1949..2152
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2103..2123
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2179..2188
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2605..2614
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2611..3318
                     /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(2756..2774)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3024..3043
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3358..3378
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site
                     "
                     /label=TVMV site
                     /translation="ETVRFQS"
     CDS             3388..3408
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site
                     "
                     /label=TVMV site
                     /translation="ETVRFQS"
     misc_feature    join(4018..4592,4593..4595,4596..4604)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4185..4202)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4412..4431
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4605..4607,4608..4925)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     polyA_signal    5767..5888
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5804..5823)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5858..5877
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5895..6350)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6032..6053)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6244..6263
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6377..6481
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6506..6526)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6550..6760
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        6572..6768
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6619..6754
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(6803..7323,1..274)
                     /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             join(6803..7323,1..274)
                     /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(6857..6876)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
       61 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
      121 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
      181 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
      241 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
      301 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
      361 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
      421 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
      481 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
      541 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
      601 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
      661 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
      721 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
      781 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
      841 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
      901 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
      961 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1021 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1081 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1141 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1201 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1261 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1321 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1381 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1441 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     1501 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     1561 gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt
     1621 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     1681 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     1741 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     1801 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     1861 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     1921 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     1981 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     2041 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     2101 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
     2161 gaaccgtcag atccgctagc caccatggcc cgtaccaagc agaccgcccg taaatccacc
     2221 ggagggaagg ctccccgcaa gcagctggcc accaaggcag ccaggaagtc cgctcctgct
     2281 accggcggag tcaagaaacc tcaccgttac cggcccggca cagtcgctct ccgcgagatc
     2341 cgccgctacc agaaatccac cgagctgctc atccgcaaac tgcctttcca gcgcctggtc
     2401 cgggagatcg ctcaggactt caagaccgac ctgcgcttcc agagctcggc cgttatggct
     2461 ctgcaggagg ccagcgaggc ttatctggtc gctctctttg aggacaccaa cctgtgcgcc
     2521 atccacgcca agagggtcac catcatgccc aaggacatcc agctggcccg cagaatccga
     2581 ggcgagaggg ctggcggacc ggtcgccacc atggtgagca agggcgagga ggataacatg
     2641 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
     2701 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     2761 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     2821 atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg
     2881 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     2941 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
     3001 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     3061 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg
     3121 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag
     3181 cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac
     3241 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     3301 atggacgagc tgtacaagta ctcagatctc gagggaggtt ccggcgggag cggaggagaa
     3361 actgtaagat tccaaagcgg tggatccgaa accgtgcgct ttcagagcgg tggcggggga
     3421 agcggcggat caggtggcgc gggaggtggc tctggaggta gcggcggcgt ggcgattctg
     3481 tggcatgaaa tgtggcatga aggcctggaa gaagcgagcc gcctgtattt tggcgaacgc
     3541 aacgtgaaag gcatgtttga agtgctggaa ccgctgcatg cgatgatgga acgcggcccg
     3601 cagaccctga aagaaaccag ctttaaccag gcgtatggcc gcgatctgat ggaagcgcag
     3661 gaatggtgcc gcaaatatat gaaaagcggc aacgtgaaag atctgaccca ggcgtgggat
     3721 ctgtattatc atgtgtttcg ccgcattggc ggcaccggcg gcagcggagg taccggcggt
     3781 agcgggggat caggtggcag cggagggacc ggtggaagcg agctcaagct tcgcggcgat
     3841 ggcgaaccga gcggcgtgcc ggtggcggtg gtgctgctgc cggtgtttgc gctgaccctg
     3901 gtggcggtgt gggcgtttgt gcgctatcgc aaacagctgt agcgctaccg gactcagatc
     3961 tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat cgatccgccc
     4021 ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
     4081 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
     4141 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
     4201 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     4261 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     4321 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     4381 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     4441 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtacac
     4501 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga
     4561 cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggtg caggtggaaa
     4621 ccattagccc gggcgatggc cgcacctttc cgaaacgcgg ccagacctgc gtggtgcatt
     4681 ataccggcat gctggaagat ggcaaaaaat ttgatagcag ccgcgatcgc aacaaaccgt
     4741 ttaaatttat gctgggcaaa caggaagtga ttcgcggctg ggaagaaggc gtggcgcaga
     4801 tgagcgtggg ccagcgcgcg aaactgacca ttagcccgga ttatgcgtat ggcgcgaccg
     4861 gccatccggg cattattccg ccgcatgcga ccctggtgtt tgatgtggaa ctgctgaaac
     4921 tggaaggcgg ctccggcggc agcggtggat caggcgggtc tggaggtagc ggcggttcaa
     4981 aggcgctact taagggagtg cgagatttta atccaatctc tgcttgcgta tgcctccttg
     5041 agaactcctc ggatgggcat agtgagagac tgtttggcat tggttttggc ccatatatca
     5101 ttgccaacca acatcttttt agaaggaaca atggagagtt gactatcaaa accatgcatg
     5161 gtgagttcaa agtcaagaac tcaacacaat tgcagatgaa accagttgag ggcagagaca
     5221 taatagttat caaaatggct aaggacttcc caccattccc tcaaaaacta aaattcagac
     5281 agcctaccat caaagataga gtgtgcatgg tatccacaaa ttttcagcag aaaagtgtct
     5341 ctagtctagt gtctgagtca tcacacattg tgcataaaga ggacacttca ttctggcaac
     5401 actggataac aacaaaggat ggacaatgtg gaagtccgct ggtttcaatc attgatggaa
     5461 atattttggg gatccacagc ctgacgcata cgaccaatgg tagcaattac ttcgtggaat
     5521 ttcctgagaa gttcgtagct acatatcttg atgccgctga tggttggtgc aagaattgga
     5581 agttcaatgc tgataagatc agttggggtt cctttattct gtgggaagat gcgccggaag
     5641 attttatgag cggcctggtg ccacgcggtg tgggtcgcga atatgtgcgc tttgcgccgg
     5701 gcagctagaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     5761 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5821 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5881 gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     5941 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     6001 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     6061 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     6121 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     6181 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     6241 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     6301 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     6361 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6421 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     6481 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     6541 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     6601 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     6661 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     6721 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     6781 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     6841 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     6901 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     6961 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     7021 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     7081 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     7141 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     7201 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     7261 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     7321 gct
//
