LOCUS       sequence-481811-        6066 bp    DNA     circular SYN 04-AUG-2025
DEFINITION  +end oriented kinesin for rapalog induced pulling experiments..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6066)
  AUTHORS   Pasolli M, Meiring JCM, Conboy JP, Koenderink GH, Akhmanova A
  TITLE     Optogenetic and chemical genetic tools for rapid repositioning of 
            vimentin intermediate filaments.
  JOURNAL   J Cell Biol. 2025 Sep 1;224(9):e202504004. doi: 
            10.1083/jcb.202504004. Epub 2025 Jul 8.
  PUBMED    40627079
REFERENCE   2  (bases 1 to 6066)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol."; date: "2025-09-1"; volume: "224(9):e202504004. doi"; pages: 
            " 10.1083/jcb.202504004. Epub 2025 Jul 8"
COMMENT     Sequence Label: HA-KIF5A-FRB
FEATURES             Location/Qualifiers
     source          1..6066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      300..888
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     789..808
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1063..1366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1367..1570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1521..1541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1618..1707
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     regulatory      1744..1753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3445..3723
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     polyA_signal    3856..3977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3893..3912)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3947..3966
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3984..4439)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4121..4142)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4333..4352
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4466..4570
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4572..4929
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4572..4763
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4595..4615)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4780..4915
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4964..5758
                     /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             4964..5758
                     /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(5018..5037)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5628..5647
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5946..5965)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5990..6037
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
       61 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
      121 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
      181 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
      241 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
      301 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
      361 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
      421 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
      481 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      541 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      601 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      661 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      721 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      781 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      841 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      901 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
      961 ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc
     1021 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     1081 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     1141 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     1201 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     1261 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     1321 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     1381 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     1441 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     1501 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     1561 aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtgcc caccatgtac
     1621 ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcc
     1681 tatccatatg acgttccaga ttacgctgga tccgtgtaca agtccggact cagatctcga
     1741 ggggccacca tggcggagac taacaacgaa tgcagcatca aggtgctttg ccgatttcgg
     1801 cccctgaacc aggccgagat tctgcggggg gacaagttca tccccatttt ccaaggggac
     1861 gacagcgtca ttattggggg aaagccatat gtctttgacc gcgtcttccc cccaaacacc
     1921 actcaggagc aggtttacca cgcctgtgcc atgcagatcg tcaaagacgt ccttgctggt
     1981 tacaatggca caatcttcgc ttatggacag acatcctcag ggaaaacgca taccatggag
     2041 gggaagctgc acgaccctca gctgatgggc atcattcccc ggatcgctcg agacatcttc
     2101 aaccacatct actccatgga tgagaacctt gaattccaca ttaaggtatc ttacttcgag
     2161 atttacctgg ataagatccg tgaccttttg gatgtgacca agacgaacct gtccgtgcat
     2221 gaggacaaaa accgggtgcc gtttgtcaag ggttgtaccg aacgctttgt gtccagccca
     2281 gaggagattc tggatgtgat cgatgagggg aagtccaacc gtcacgtagc tgtcaccaac
     2341 atgaacgagc acagttctcg gagccacagc atcttcctca tcaacatcaa gcaggagaac
     2401 gtagagaccg agcagaagct cagcgggaag ctgtacctcg tggatctggc cggaagcgag
     2461 aaggtcagca agacaggggc agagggagcc gttctggacg aggcaaagaa tatcaacaag
     2521 tcgctgtcgg ccctggggaa cgtgatctct gcactggcag agggcaccaa aagctacgtg
     2581 ccgtaccgcg acagcaaaat gacgaggatt ctccaggact ctctgggagg gaactgcagg
     2641 actaccatgt tcatctgctg ctcgccgtcc agctacaatg acgcagagac caagtccacg
     2701 ctcatgtttg gacagcgggc gaagaccatc aagaacactg cctcagtgaa tctggagctg
     2761 actgctgagc agtggaagaa gaagtatgag aaggagaagg agaagaccaa ggcccagaag
     2821 gagacaattg cgaagctaga ggctgagctt agccggtggc gcaatggaga gaatgtgcct
     2881 gagactgagc gcctggctgg agaggactca gctctgggag ctgagctctg cgaggagacc
     2941 cctgtgaatg acaactcatc cattgtggta cgcatcgcac ctgaggaaag gcagaaatat
     3001 gaggaagaga tccgccgtct ctacaagcag cttgatgaca aggatgatga gatcaaccag
     3061 cagagccagc tcattgagaa gctgaagcag cagatgctgg accaggaaga gctgctcgtg
     3121 tccactcggg gagacaacga gaaggtccag cgggagctta gccacctgca gtccgagaac
     3181 gatgctgcga aggacgaggt gaaggaagtg ctgcaggccc tagaggagct ggcggtcaac
     3241 tacgaccaga agtcccagga ggtggaggag aagagccagc agaaccagct gctggtggac
     3301 gagctgtccc agaaagtggc caccatgctg tccctggagt ccgagctaca gcggctccag
     3361 gaggtcagtg gacaccagcg aaagcggatc gctgaggtgc tgaatgggct gatgagggac
     3421 ctgagtggta gcggcagcgg tagcatcctc tggcatgaga tgtggcatga aggcctggaa
     3481 gaggcatctc gtttgtactt tggggaaagg aacgtgaaag gcatgtttga ggtgctggag
     3541 cccttgcatg ctatgatgga acggggcccc cagactctga aggaaacatc ctttaatcag
     3601 gcctatggtc gagatttaat ggaggcccaa gagtggtgca ggaagtacat gaaatcaggg
     3661 aatgtcaagg acctcctcca agcctgggac ctctattatc atgtgttccg acgaatctca
     3721 aagtagggat ccaccggatc tagataactg atcataatca gccataccac atttgtagag
     3781 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     3841 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3901 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3961 ctcatcaatg tatcttaacg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     4021 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     4081 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     4141 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     4201 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     4261 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     4321 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     4381 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     4441 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4501 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4561 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     4621 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4681 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4741 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4801 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4861 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4921 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     4981 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     5041 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     5101 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     5161 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     5221 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     5281 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     5341 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     5401 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     5461 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     5521 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     5581 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     5641 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     5701 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     5761 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     5821 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     5881 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     5941 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     6001 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     6061 ggctgg
//
