LOCUS       sequence-313608-        8020 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 8020)
  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 8020)
  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..8020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             67..90
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             133..306
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAYYEILHLPNLNEGQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             307..480
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAEKLNDAQAPK"
     CDS             538..558
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             580..600
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2287..3000
                     /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             2287..3000
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2317..2345)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2940..2960
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3007..3552
                     /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    3686..3820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3723..3742)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3777..3796
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  3941..4106
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     misc_recomb     4113..4818
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4201..4573
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4230..4818)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     rep_origin      5008..5596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5497..5516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_recomb     5852..6679
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             5852..5976
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6014..6619)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     mobile_element  6708..6931
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(6998..7531)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7720..7725,7726..7742,7743..7748))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    complement(7819..7852)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        7929..8020
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     7945..7962
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
ORIGIN
        1 attccggatt attcataccg tcccaccatc gggcgcggat ctcggtccga aaccatgtcg
       61 tactaccatc accatcacca tcaccatcac ggttctatgt gcatggcagg ccttgcgcaa
      121 cacgatgaag ccgtggacaa caaattcaac aaagaacaac aaaacgcgta ctatgagatc
      181 ttacatttac ctaacttaaa cgaaggacaa cgaaacgcct tcatccaaag tttaaaagat
      241 gacccaagcc aaagcgctaa ccttttagca gaagctaaaa agctaaatga tgctcaggcg
      301 ccgaaagtag acaacaaatt caacaaagaa caacaaaacg cgttctatga gatcttacat
      361 ttacctaact taaacgaaga acaacgaaac gccttcatcc aaagtttaaa agatgaccca
      421 agccaaagcg ctaacctttt agcagaagct gaaaagctaa atgatgctca ggcgccgaaa
      481 gtagacgcaa attccgcggg gaagtcaacc gattatgata ttccaactac tgctagcgag
      541 aacctatatt tccagggaga ttatgatatc cctacgactg agaatcttta ttttcagggc
      601 atggcggacc tggccgagtg caacatcaaa gtgatgtgtc gcttcagacc tctcaacgag
      661 tctgaagtga accgcggcga caagtacatc gccaagtttc agggagaaga cacggtcgtg
      721 atcgcgtcca agccttatgc atttgatcgg gtgttccagt caagcacatc tcaagagcaa
      781 gtgtataatg actgtgcaaa gaagattgtt aaagatgtac ttgaaggata taatggaaca
      841 atatttgcat atggacaaac atcctctggg aagacacaca caatggaggg taaacttcat
      901 gatccagaag gcatgggaat tattccaaga atagtgcaag atatttttaa ttatatttac
      961 tccatggatg aaaatttgga atttcatatt aaggtttcat attttgaaat atatttggat
     1021 aagataaggg acctgttaga tgtttcaaag accaaccttt cagttcatga agacaaaaac
     1081 cgagttccct atgtaaaggg gtgcacagag cgttttgtat gtagtccaga tgaagttatg
     1141 gataccatag atgaaggaaa atccaacaga catgtagcag ttacaaatat gaatgaacat
     1201 agctctagga gtcacagtat atttcttatt aatgtcaaac aagagaacac acaaacggaa
     1261 caaaagctga gtggaaaact ttatctggtt gatttagctg gtagtgaaaa ggttagtaaa
     1321 actggagctg aaggtgctgt gctggatgaa gctaaaaaca tcaacaagtc actttctgct
     1381 cttggaaatg ttatttctgc tttggctgag ggtagtacat atgttccata tcgagatagt
     1441 aaaatgacaa gaatccttca agattcatta ggtggcaact gtagaaccac tattgtaatt
     1501 tgctgctctc catcatcata caatgagtct gaaacaaaat ctacactctt atttggccaa
     1561 agggccaaaa caattaagaa cacagtttgt gtcaatgtgg agttaactgc agaacagtgg
     1621 aaaaagaagt atgaaaaaga aaaagaaaaa aataagatcc tgcggaacac tattcagtgg
     1681 cttgaaaatg agctcaacag atggcgtaat ggggagacgg tgcctattga tgaacagttt
     1741 gacaaagaga aagccaactt ggaagctttc acagtggata aagatattac tcttaccaat
     1801 gataaaccag caaccgcaat tggagttata ggaaatttta ctgatgctga aagaagaaag
     1861 tgtgaagaag aaattgctaa attatacaaa cagcttgatg acaaggatga agaaattaac
     1921 cagcaaagtc aactggtaga gaaactgaag acgcaaatgt tggatcagga ggagcttttg
     1981 gcatctacca gaagggatca agacaatatg caagctgagc tgaatcgcct tcaagcagaa
     2041 aatgatgcct ctaaagaaga agtgaaagaa gttttacagg ccctagaaga acttgctgtc
     2101 aattatgatc agaagtctca ggaagttgaa gacaaaacta aggaatatga attgcttagt
     2161 gatgaattga atcagaaatc ggcaacttta gcgagtatag atgctgagct tcagaaactt
     2221 aaggaaatga ccaaccacca gaaaaaacga gcagctgaga tgatggcatc tttactaaaa
     2281 ggttctatga gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta
     2341 gatggtgatg ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca
     2401 tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca
     2461 acacttgtca ctactctgac ttatggtgtt caatgctttt caagataccc agatcatatg
     2521 aaacagcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata
     2581 tttttcaaag atgacgggaa ctacaagaca cgtgctgaag tcaagtttga aggtgatacc
     2641 cttgttaata gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga
     2701 cacaaattgg aatacaacta taactcacac aatgtataca tcatggcaga caaacaaaag
     2761 aatggaatca aagttaactt caaaattaga cacaacattg aagatggaag cgttcaacta
     2821 gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac
     2881 cattacctgt ccacacaatc tgccctttcg aaagatccca acgaaaagag agaccacatg
     2941 gtccttcttg agtttgtaac agctgctggg attacacatg gcatggatga actatacaaa
     3001 ggttctatgg acaaagactg cgaaatgaag cgcaccaccc tggatagccc tctgggcaag
     3061 ctggaactgt ctgggtgcga acagggcctg caccgtatca tcttcctggg caaaggaaca
     3121 tctgccgccg acgccgtgga agtgcctgcc ccagccgccg tgctgggcgg accagagcca
     3181 ctgatgcagg ccaccgcctg gctcaacgcc tactttcacc agcctgaggc catcgaggag
     3241 ttccctgtgc cagccctgca ccacccagtg ttccagcagg agagctttac ccgccaggtg
     3301 ctgtggaaac tgctgaaagt ggtgaagttc ggagaggtca tcagctacag ccacctggcc
     3361 gccctggccg gcaatcccgc cgccaccgcc gccgtgaaaa ccgccctgag cggaaatccc
     3421 gtgcccattc tgatcccctg ccacctggtg gtgcagggcg acctggacgt ggggggctac
     3481 gagggcgggc tcgccgtgaa agagtggctg ctggcccacg agggccacag actgggcaag
     3541 cctgggctgg gttaatgagc ttgtcgagaa gtactagagg atcataatca gccataccac
     3601 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     3661 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     3721 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3781 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgatcactgc ttgagcctag
     3841 aagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     3901 aataactatc ataaccccta gggtataccc atctaattgg aaccagataa gtgaaatcta
     3961 gttccaaact attttgtcat ttttaatttt cgtattagct tacgacgcta cacccagttc
     4021 ccatctattt tgtcactctt ccctaaataa tccttaaaaa ctccatttcc acccctccca
     4081 gttcccaact attttgtccg cccacaaccg gtggaggaaa ttctccttga agtttccctg
     4141 gtgttcaaag taaaggagtt tgcaccagac gcacctctgt tcactggtcc ggcgtattaa
     4201 aacacgatac attgttatta gtacatttat taagcgctag attctgtgcg ttgttgattt
     4261 acagacaatt gttgtacgta ttttaataat tcattaaatt tataatcttt agggtggtat
     4321 gttagagcga aaatcaaatg attttcagcg tctttatatc tgaatttaaa tattaaatcc
     4381 tcaatagatt tgtaaaatag gtttcgatta gtttcaaaca agggttgttt ttccgaaccg
     4441 atggctggac tatctaatgg attttcgctc aacgccacaa aacttgccaa atcttgtagc
     4501 agcaatctag ctttgtcgat attcgtttgt gttttgtttt gtaataaagg ttcgacgtcg
     4561 ttcaaaatat tatgcgcttt tgtatttctt tcatcactgt cgttagtgta caattgactc
     4621 gacgtaaaca cgttaaatag agcttggaca tatttaacat cgggcgtgtt agctttatta
     4681 ggccgattat cgtcgtcgtc ccaaccctcg tcgttagaag ttgcttccga agacgatttt
     4741 gccatagcca cacgacgcct attaattgtg tcggctaaca cgtccgcgat caaatttgta
     4801 gttgagcttt ttggaattac cggttgactt gggtcaactg tcagaccaag tttactcata
     4861 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4921 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4981 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5041 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5101 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5161 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5221 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5281 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5341 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5401 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5461 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5521 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5581 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5641 gccttttgct cacatgttct ttcctgcgtt atcccctgat tgacttgggt cgctcttcct
     5701 gtggatgcgc aggtatgtac aggaagaggt ttatactaaa ctgttacatt gcaaacgtgg
     5761 tttcgtgtgc caagtgtgaa aaccgatgtt taatcaaggc tctgacgcat ttctacaacc
     5821 acgactctaa gtgtgtgggt gaagtcatgc atgcatcttt taatcaaatc ccaagatgtg
     5881 tataaaccac caaactgcca aaaaatgaaa actgtcgaca agctctgtcc gtttgctggc
     5941 aactgcaagg gtctcaatcc tatttgtaat tattgaataa taaaacaatt ataaatgtca
     6001 aatttgtttt ttattaacga tacaaaccaa acgcaacaag aacatttgta gtattatcta
     6061 taattgaaaa cgcgtagtta taatcgctga ggtaatattt aaaatcattt tcaaatgatt
     6121 cacagttaat ttgcgacaat ataattttat tttcacataa actagacgcc ttgtcgtctt
     6181 cttcttcgta ttccttctct ttttcatttt tctcttcata aaaattaaca tagttattat
     6241 cgtatccata tatgtatcta tcgtatagag taaatttttt gttgtcataa atatatatgt
     6301 cttttttaat ggggtgtata gtaccgctgc gcatagtttt tctgtaattt acaacagtgc
     6361 tattttctgg tagttcttcg gagtgtgttg ctttaattat taaatttata taatcaatga
     6421 atttgggatc gtcggttttg tacaatatgt tgccggcata gtacgcagct tcttctagtt
     6481 caattacacc attttttagc agcaccggat taacataact ttccaaaatg ttgtacgaac
     6541 cgttaaacaa aaacagttca cctccctttt ctatactatt gtctgcgagc agttgtttgt
     6601 tgttaaaaat aacagccatt gtaatgagac gcacaaacta atatcacaaa ctggaaatgt
     6661 ctatcaatat atagttgctg attgcgcaga tgccctgcgt aagcgggtgt gggcggacaa
     6721 taaagtctta aactgaacaa aatagatcta aactatgaca ataaagtctt aaactagaca
     6781 gaatagttgt aaactgaaat cagtccagtt atgctgtgaa aaagcatact ggacttttgt
     6841 tatggctaaa gcaaactctt cattttctga agtgcaaatt gcccgtcgta ttaaagaggg
     6901 gcgtggccaa gggcatgtaa agactatatt cgcggcgttg tgacaattta ccgaacaact
     6961 ccgcggccgg gaagccgatc tcggcttgaa cgaattgtta ggtggcggta cttgggtcga
     7021 tatcaaagtg catcacttct tcccgtatgc ccaactttgt atagagagcc actgcgggat
     7081 cgtcaccgta atctgcttgc acgtagatca cataagcacc aagcgcgttg gcctcatgct
     7141 tgaggagatt gatgagcgcg gtggcaatgc cctgcctccg gtgctcgccg gagactgcga
     7201 gatcatagat atagatctca ctacgcggct gctcaaactt gggcagaacg taagccgcga
     7261 gagcgccaac aaccgcttct tggtcgaagg cagcaagcgc gatgaatgtc ttactacgga
     7321 gcaagttccc gaggtaatcg gagtccggct gatgttggga gtaggtggct acgtctccga
     7381 actcacgacc gaaaagatca agagcagccc gcatggattt gacttggtca gggccgagcc
     7441 tacatgtgcg aatgatgccc atacttgagc cacctaactt tgttttaggg cgactgccct
     7501 gctgcgtaac atcgttgctg ctgcgtaaca tcgttgctgc tccataacat caaacatcga
     7561 cccacggcgt aacgcgcttg ctgcttggat gcccgaggca tagactgtac aaaaaaacag
     7621 tcataacaag ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt
     7681 ctggaccagt tgcgtgagcg catacgctac ttgcattaca gtttacgaac cgaacaggct
     7741 tatgtcaact gggttcgtgc cttcatccgt ttccacggtg tgcgtcaccc ggcaaccttg
     7801 ggcagcagcg aagtcgccat aacttcgtat agcatacatt atacgaagtt atctgtaact
     7861 ataacggtcc taaggtagcg agtttaaaca ctagtatcga ttcgcgacct actccggaat
     7921 attaatagat catggagata attaaaatga taaccatctc gcaaataaat aagtatttta
     7981 ctgttttcgt aacagttttg taataaaaaa acctataaat
//
