LOCUS       sequence-308978-        8016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses kinesin-1 (amino acids 1-560) in Sf9 cells with a 
            C-terminal GFP and SNAP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8016)
  AUTHORS   Ferro LS, Fang Q, Eshun-Wilson L, Fernandes J, Jack A, Farrell DP, 
            Golcuk M, Huijben T, Costa K, Gur M, DiMaio F, Nogales E, Yildiz A
  TITLE     Structural and functional insight into regulation of kinesin-1 by 
            microtubule-associated protein MAP7.
  JOURNAL   Science. 2022 Jan 21;375(6578):326-331. doi: 
            10.1126/science.abf6154. Epub 2022 Jan 20.
  PUBMED    35050657
REFERENCE   2  (bases 1 to 8016)
  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: 
            "10.1126/science.abf6154"; journalName: "Science"; date: 
            "2022-01-21- 21"; volume: "375"; issue: "6578"; pages: "326-331"
COMMENT     Sequence Label: pOmnibac_ZZ_linker_K560_GFP_SNAP
FEATURES             Location/Qualifiers
     source          1..8016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_recomb     457..1284
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             457..581
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(619..1224)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     mobile_element  1313..1536
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(1603..2136)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(2325..2330,2331..2347,2348..2353))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    complement(2424..2457)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2534..2625
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     2550..2567
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             2692..2715
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2758..2931
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             2932..3105
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             3163..3183
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3205..3225
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4912..5625
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             4912..5625
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4942..4970)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5565..5585
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             5632..6177
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHLVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     polyA_signal    6311..6445
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6348..6367)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6402..6421
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  6566..6731
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     misc_recomb     6738..7443
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           6826..7198
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(6855..7443)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     rep_origin      join(7633..8016,1..205)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
       61 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
      121 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
      181 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
      241 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt gacttgggtc
      301 gctcttcctg tggatgcgca ggtatgtaca ggaagaggtt tatactaaac tgttacattg
      361 caaacgtggt ttcgtgtgcc aagtgtgaaa accgatgttt aatcaaggct ctgacgcatt
      421 tctacaacca cgactctaag tgtgtgggtg aagtcatgca tcttttaatc aaatcccaag
      481 atgtgtataa accaccaaac tgccaaaaaa tgaaaactgt cgacaagctc tgtccgtttg
      541 ctggcaactg caagggtctc aatcctattt gtaattattg aataataaaa caattataaa
      601 tgtcaaattt gttttttatt aacgatacaa accaaacgca acaagaacat ttgtagtatt
      661 atctataatt gaaaacgcgt agttataatc gctgaggtaa tatttaaaat cattttcaaa
      721 tgattcacag ttaatttgcg acaatataat tttattttca cataaactag acgccttgtc
      781 gtcttcttct tcgtattcct tctctttttc atttttctct tcataaaaat taacatagtt
      841 attatcgtat ccatatatgt atctatcgta tagagtaaat tttttgttgt cataaatata
      901 tatgtctttt ttaatggggt gtatagtacc gctgcgcata gtttttctgt aatttacaac
      961 agtgctattt tctggtagtt cttcggagtg tgttgcttta attattaaat ttatataatc
     1021 aatgaatttg ggatcgtcgg ttttgtacaa tatgttgccg gcatagtacg cagcttcttc
     1081 tagttcaatt acaccatttt ttagcagcac cggattaaca taactttcca aaatgttgta
     1141 cgaaccgtta aacaaaaaca gttcacctcc cttttctata ctattgtctg cgagcagttg
     1201 tttgttgtta aaaataacag ccattgtaat gagacgcaca aactaatatc acaaactgga
     1261 aatgtctatc aatatatagt tgctgattgc gcagatgccc tgcgtaagcg ggtgtgggcg
     1321 gacaataaag tcttaaactg aacaaaatag atctaaacta tgacaataaa gtcttaaact
     1381 agacagaata gttgtaaact gaaatcagtc cagttatgct gtgaaaaagc atactggact
     1441 tttgttatgg ctaaagcaaa ctcttcattt tctgaagtgc aaattgcccg tcgtattaaa
     1501 gaggggcgtg gccaagggca tgtaaagact atattcgcgg cgttgtgaca atttaccgaa
     1561 caactccgcg gccgggaagc cgatctcggc ttgaacgaat tgttaggtgg cggtacttgg
     1621 gtcgatatca aagtgcatca cttcttcccg tatgcccaac tttgtataga gagccactgc
     1681 gggatcgtca ccgtaatctg cttgcacgta gatcacataa gcaccaagcg cgttggcctc
     1741 atgcttgagg agattgatga gcgcggtggc aatgccctgc ctccggtgct cgccggagac
     1801 tgcgagatca tagatataga tctcactacg cggctgctca aacttgggca gaacgtaagc
     1861 cgcgagagcg ccaacaaccg cttcttggtc gaaggcagca agcgcgatga atgtcttact
     1921 acggagcaag ttcccgaggt aatcggagtc cggctgatgt tgggagtagg tggctacgtc
     1981 tccgaactca cgaccgaaaa gatcaagagc agcccgcatg gatttgactt ggtcagggcc
     2041 gagcctacat gtgcgaatga tgcccatact tgagccacct aactttgttt tagggcgact
     2101 gccctgctgc gtaacatcgt tgctgctgcg taacatcgtt gctgctccat aacatcaaac
     2161 atcgacccac ggcgtaacgc gcttgctgct tggatgcccg aggcatagac tgtacaaaaa
     2221 aacagtcata acaagccatg aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca
     2281 aggttctgga ccagttgcgt gagcgcatac gctacttgca ttacagttta cgaaccgaac
     2341 aggcttatgt caactgggtt cgtgccttca tccgtttcca cggtgtgcgt cacccggcaa
     2401 ccttgggcag cagcgaagtc gccataactt cgtatagcat acattatacg aagttatctg
     2461 taactataac ggtcctaagg tagcgagttt aaacactagt atcgattcgc gacctactcc
     2521 ggaatattaa tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta
     2581 ttttactgtt ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca
     2641 taccgtccca ccatcgggcg cggatctcgg tccgaaacca tgtcgtacta ccatcaccat
     2701 caccatcacc atcacggttc tatgtgcatg gcaggccttg cgcaacacga tgaagccgtg
     2761 gacaacaaat tcaacaaaga acaacaaaac gcgttctatg agatcttaca tttacctaac
     2821 ttaaacgaag aacaacgaaa cgccttcatc caaagtttaa aagatgaccc aagccaaagc
     2881 gctaaccttt tagcagaagc taaaaagcta aatgatgctc aggcgccgaa agtagacaac
     2941 aaattcaaca aagaacaaca aaacgcgttc tatgagatct tacatttacc taacttaaac
     3001 gaagaacaac gaaacgcctt catccaaagt ttaaaagatg acccaagcca aagcgctaac
     3061 cttttagcag aagctaaaaa gctaaatgat gctcaggcgc cgaaagtaga cgcaaattcc
     3121 gcggggaagt caaccgatta tgatattcca actactgcta gcgagaacct atatttccag
     3181 ggagattatg atatccctac gactgagaat ctttattttc agggcatggc ggacctggcc
     3241 gagtgcaaca tcaaagtgat gtgtcgcttc agacctctca acgagtctga agtgaaccgc
     3301 ggcgacaagt acatcgccaa gtttcaggga gaagacacgg tcgtgatcgc gtccaagcct
     3361 tatgcatttg atcgggtgtt ccagtcaagc acatctcaag agcaagtgta taatgactgt
     3421 gcaaagaaga ttgttaaaga tgtacttgaa ggatataatg gaacaatatt tgcatatgga
     3481 caaacatcct ctgggaagac acacacaatg gagggtaaac ttcatgatcc agaaggcatg
     3541 ggaattattc caagaatagt gcaagatatt tttaattata tttactccat ggatgaaaat
     3601 ttggaatttc atattaaggt ttcatatttt gaaatatatt tggataagat aagggacctg
     3661 ttagatgttt caaagaccaa cctttcagtt catgaagaca aaaaccgagt tccctatgta
     3721 aaggggtgca cagagcgttt tgtatgtagt ccagatgaag ttatggatac catagatgaa
     3781 ggaaaatcca acagacatgt agcagttaca aatatgaatg aacatagctc taggagtcac
     3841 agtatatttc ttattaatgt caaacaagag aacacacaaa cggaacaaaa gctgagtgga
     3901 aaactttatc tggttgattt agctggtagt gaaaaggtta gtaaaactgg agctgaaggt
     3961 gctgtgctgg atgaagctaa aaacatcaac aagtcacttt ctgctcttgg aaatgttatt
     4021 tctgctttgg ctgagggtag tacatatgtt ccatatcgag atagtaaaat gacaagaatc
     4081 cttcaagatt cattaggtgg caactgtaga accactattg taatttgctg ctctccatca
     4141 tcatacaatg agtctgaaac aaaatctaca ctcttatttg gccaaagggc caaaacaatt
     4201 aagaacacag tttgtgtcaa tgtggagtta actgcagaac agtggaaaaa gaagtatgaa
     4261 aaagaaaaag aaaaaaataa gatcctgcgg aacactattc agtggcttga aaatgagctc
     4321 aacagatggc gtaatgggga gacggtgcct attgatgaac agtttgacaa agagaaagcc
     4381 aacttggaag ctttcacagt ggataaagat attactctta ccaatgataa accagcaacc
     4441 gcaattggag ttataggaaa ttttactgat gctgaaagaa gaaagtgtga agaagaaatt
     4501 gctaaattat acaaacagct tgatgacaag gatgaagaaa ttaaccagca aagtcaactg
     4561 gtagagaaac tgaagacgca aatgttggat caggaggagc ttttggcatc taccagaagg
     4621 gatcaagaca atatgcaagc tgagctgaat cgccttcaag cagaaaatga tgcctctaaa
     4681 gaagaagtga aagaagtttt acaggcccta gaagaacttg ctgtcaatta tgatcagaag
     4741 tctcaggaag ttgaagacaa aactaaggaa tatgaattgc ttagtgatga attgaatcag
     4801 aaatcggcaa ctttagcgag tatagatgct gagcttcaga aacttaagga aatgaccaac
     4861 caccagaaaa aacgagcagc tgagatgatg gcatctttac taaaaggttc tatgagtaaa
     4921 ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat
     4981 gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc
     5041 cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact
     5101 ctgacttatg gtgttcaatg cttttcaaga tacccagatc atatgaaaca gcatgacttt
     5161 ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac
     5221 gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc
     5281 gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac
     5341 aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagtt
     5401 aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa
     5461 caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca
     5521 caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt
     5581 gtaacagctg ctgggattac acatggcatg gatgaactat acaaaggttc tatggacaaa
     5641 gactgcgaaa tgaagcgcac caccctggat agccctctgg gcaagctgga actgtctggg
     5701 tgcgaacagg gcctgcaccg tatcatcttc ctgggcaaag gaacatctgc cgccgacgcc
     5761 gtggaagtgc ctgccccagc cgccgtgctg ggcggaccag agccactgat gcaggccacc
     5821 gcctggctca acgcctactt tcaccagcct gaggccatcg aggagttccc tgtgccagcc
     5881 ctgcaccacc cagtgttcca gcaggagagc tttacccgcc aggtgctgtg gaaactgctg
     5941 aaagtggtga agttcggaga ggtcatcagc tacagccacc tggccgccct ggccggcaat
     6001 cccgccgcca ccgccgccgt gaaaaccgcc ctgagcggaa atcccgtgcc cattctgatc
     6061 ccctgccacc tggtggtgca gggcgacctg gacgtggggg gctacgaggg cgggctcgcc
     6121 gtgaaagagt ggctgctggc ccacgagggc cacagactgg gcaagcctgg gctgggttaa
     6181 tgagcttgtc gagaagtact agaggatcat aatcagccat accacatttg tagaggtttt
     6241 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     6301 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     6361 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     6421 caatgtatct tatcatgtct ggatctgatc actgcttgag cctagaagat ccggctgcta
     6481 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctatcataac
     6541 ccctagggta tacccatcta attggaacca gataagtgaa atctagttcc aaactatttt
     6601 gtcattttta attttcgtat tagcttacga cgctacaccc agttcccatc tattttgtca
     6661 ctcttcccta aataatcctt aaaaactcca tttccacccc tcccagttcc caactatttt
     6721 gtccgcccac aaccggtgga ggaaattctc cttgaagttt ccctggtgtt caaagtaaag
     6781 gagtttgcac cagacgcacc tctgttcact ggtccggcgt attaaaacac gatacattgt
     6841 tattagtaca tttattaagc gctagattct gtgcgttgtt gatttacaga caattgttgt
     6901 acgtatttta ataattcatt aaatttataa tctttagggt ggtatgttag agcgaaaatc
     6961 aaatgatttt cagcgtcttt atatctgaat ttaaatatta aatcctcaat agatttgtaa
     7021 aataggtttc gattagtttc aaacaagggt tgtttttccg aaccgatggc tggactatct
     7081 aatggatttt cgctcaacgc cacaaaactt gccaaatctt gtagcagcaa tctagctttg
     7141 tcgatattcg tttgtgtttt gttttgtaat aaaggttcga cgtcgttcaa aatattatgc
     7201 gcttttgtat ttctttcatc actgtcgtta gtgtacaatt gactcgacgt aaacacgtta
     7261 aatagagctt ggacatattt aacatcgggc gtgttagctt tattaggccg attatcgtcg
     7321 tcgtcccaac cctcgtcgtt agaagttgct tccgaagacg attttgccat agccacacga
     7381 cgcctattaa ttgtgtcggc taacacgtcc gcgatcaaat ttgtagttga gctttttgga
     7441 attaccggtt gacttgggtc aactgtcaga ccaagtttac tcatatatac tttagattga
     7501 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     7561 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7621 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     7681 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     7741 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     7801 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7861 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7921 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7981 ggagcgaacg acctacaccg aactgagata cctaca
//
