LOCUS       sequence-425805-        6790 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses fusion of Kinesin heavy chain 5A (1-572) with GFP and SspB
            (micro).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6790)
  AUTHORS   Liu X, Xu L, Song Y, Zhao Z, Li X, Wong CY, Chen R, Feng J, Gou Y, 
            Qi Y, Chow HM, Yao S, Wang Y, Gao S, Liu X, Duan L
  TITLE     Force-induced tail-autotomy mitochondrial fission and biogenesis of 
            matrix-excluded mitochondrial-derived vesicles for quality control.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Apr 2;121(14):e2217019121. doi: 
            10.1073/pnas.2217019121. Epub 2024 Mar 28.
  PUBMED    38547062
REFERENCE   2  (bases 1 to 6790)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-04-2"; volume: "121(14):e2217019121. 
            doi"; pages: " 10.1073/pnas.2217019121. Epub 2024 Mar 28"
COMMENT     Sequence Label: KIF5A-GFP-SspB
FEATURES             Location/Qualifiers
     source          1..6790
                     /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      606..615
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2361..2370
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2367..2369,2370..2372,2373..3083)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2367..2369,2370..2372,2373..3083)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2412..2433)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2673..2692)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3020..3041
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(3120..3326,3327..3329,3330..3449)
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein and with the mutation R74Q to reduce the 
                     affinity for iLID"
                     /label=SspB micro
                     /note="dimerizes with iLID at moderate affinity to create 
                     iLID micro (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNAQFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     polyA_signal    3578..3699
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3615..3634)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3669..3688
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3706..4161)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3843..3864)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4055..4074
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4188..4292
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4294..4651
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4294..4485
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4317..4337)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4502..4637
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4686..5480
                     /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             4686..5480
                     /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(4740..4759)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5350..5369
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5668..5687)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5712..5759
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6088..6676
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6577..6596
                     /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 agcttgccac catgggcgcg cctgaattcg cggagaccaa taacgaatgc agcatcaagg
      661 tgctttgccg atttcggccc ctgaaccagg ccgagatcct gcggggggac aagttcatcc
      721 ccattttcca aggggacgac agcgtcatta ttgggggaaa gccatatgtc ttcgaccgtg
      781 tattcccccc aaacacgact caggagcagg tttaccacgc ctgtgccatg cagatcgtca
      841 aagacgtcct tgctggctac aacggcacaa tttttgctta tggacagaca tcctcaggga
      901 aaacgcacac catggaggga aagctgcacg accctcagct catgggcatc attccccgga
      961 tcgcccgaga catcttcaac cacatctact ccatggatga gaaccttgag ttccacatta
     1021 aggtgtctta ctttgagatt tacctggata agatccgtga ccttttggat gtgaccaaga
     1081 caaacctgtc tgtgcatgag gacaaaaacc gggtgccgtt tgtcaagggt tgtaccgaac
     1141 gctttgtgtc cagcccagag gagattctgg atgtgattga tgaggggaag tcgaaccgtc
     1201 acgttgccgt caccaacatg aatgagcaca gctctcggag tcacagcatc ttcctcatca
     1261 acatcaagca ggagaacata gagacggagc agaagctcag cgggaaactg taccttgtgg
     1321 atctggccgg aagcgagaag gtcagcaaga caggagcaga gggagccgtt ctggatgagg
     1381 caaagaatat caataagtcg ctgtcggccc tggggaatgt gatctctgct ctggccgagg
     1441 gcactaaaag ctacgtgccg taccgtgaca gcaaaatgac gaggattctc caggactctc
     1501 tgggagggaa ctgcaggacc accatgttca tctgctgctc accatccagc tacaatgacg
     1561 cagagaccaa gtccacactc atgtttgggc agcgggcaaa gaccatcaag aacactgcct
     1621 cagtgaatct ggagttgact gctgagcaat ggaagaagaa atatgagaag gagaaggaga
     1681 agaccaaggc ccagaaggag acaattgcga agctggaggc tgagcttagc cggtggcgca
     1741 atggagagaa tgtgcctgag actgagcgcc tggctggaga ggactcagct ctggcagccg
     1801 agatctgtga ggagacccct gtgaatgaca actcatccat cgtggtacgc atcgcaccgg
     1861 aggaaaggca gaaatacgag gaagagatcc gccgtctcta caagcagctc gacgacaagg
     1921 atgatgagat caaccagcag agccaactca ttgagaagct gaagcagcag atgctggacc
     1981 aggaagagct gctagtgtcc actcggggag acaatgagaa ggtccagcgg gagctgagcc
     2041 acctgcagtc tgagaacgat gccgcgaagg aggaggtgaa ggaagtgttg caggccctgg
     2101 aggagctggc agtcaactat gaccagaagt cccaggaggt ggaggagaag agccagcaga
     2161 accagctgct ggtggatgag ctgtcccaga aagtggccac catgctgtcc ctggagtctg
     2221 agctacagcg gctccaggaa gtcagtggac accagcgaaa acggattgct gaggtgctga
     2281 atggactgat gaaggacctg agtgagttca gtgtcatcgt gggcaagtcg acggtaccgc
     2341 gggcccggga tccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     2401 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2461 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2521 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2581 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2641 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2701 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2761 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2821 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2881 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2941 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     3001 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3061 tcggcatgga cgagctgtac aagggcattg atctgagcgg cctgaccctg caggaattca
     3121 gctccccgaa acgccctaag ctgctgcgtg aatattacga ttggctggtt gataacagct
     3181 ttaccccata tctggtggtg gatgccacat acctgggcgt gaacgtgccc gtggagtatg
     3241 tgaaagacgg tcagatcgtg ctgaatctgt ctgcaagtgc gaccggcaac ctgcaactga
     3301 caaatgattt tatccagttc aacgcccagt ttaagggcgt gtctcgtgaa ctgtatatcc
     3361 cgatgggtgc cgctctggcc atttacgctc gcgagaacgg cgatggtgtg atgttcgaac
     3421 cagaagaaat ctatgacgag ctgaatattg gttaaagcgg ccgcgactct agatcataat
     3481 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     3541 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     3601 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3661 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     3721 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3781 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3841 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3901 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3961 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     4021 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     4081 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     4141 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4201 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4261 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     4321 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4381 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4441 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4501 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4561 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     4621 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     4681 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     4741 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     4801 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     4861 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     4921 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     4981 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     5041 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     5101 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     5161 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     5221 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     5281 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     5341 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     5401 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     5461 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     5521 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     5581 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     5641 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     5701 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     5761 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     5821 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     5881 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     5941 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6001 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6061 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6121 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6181 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     6241 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6301 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6361 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6421 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6481 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6541 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6601 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6661 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6721 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     6781 cgccatgcat
//
