LOCUS       sequence-263583-        7067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Drosophila Kinesin-1 truncated at 560 and biotinylated at 
            tail for single-molecule assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7067)
  AUTHORS   Hancock
  TITLE     Hancock Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7067)
  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: 
            "Unpublished"
COMMENT     Sequence Label: K560_AviC_coBirA
FEATURES             Location/Qualifiers
     source          1..7067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             1542..1550
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1705..1722
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1729..1773
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1774..1791
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1854..2507
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2465..2487
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2514..2531
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2532..3494
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2532..3491
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             3518..3535
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3588..3606)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3602..3649
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3686..4141
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3773..3792)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3983..4004
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4167..4271
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4272..4340,4341..5132)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4490..4509)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5303..5891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5792..5811
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6045..6062
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6077..6216
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6142..6161)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6302..6324
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6318..6509)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     7018..7037
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7018..7036
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tttgtttaac tttaagaagg agatatacat atgagcgcag aacgagaaat tccagccgag
       61 gacagcatca aagtggtctg ccgattccga ccgctgaacg acagcgaaga gaaggccggc
      121 tccaagttcg tggtcaagtt ccccaacaat gtggaggaga actgcatatc catagcgggc
      181 aaggtgtatt tgttcgacaa ggtcttcaaa ccgaatgcat cccaggaaaa ggtctacaat
      241 gaggcggcca agtccattgt tacggatgtc ctggccgggt acaatggaac gatattcgca
      301 tacggtcaga cgtcctccgg aaaaacgcat acgatggagg gcgtgatcgg ggactccgta
      361 aaacagggta tcatacctag gatcgtcaac gacattttca atcacatcta cgcgatggag
      421 gtgaacctgg agtttcacat caaggtctcc tactacgaga tctacatgga caagattcga
      481 gatctgttgg acgtctccaa ggtgaacctt agtgtgcacg aggataagaa ccgggtgccg
      541 tacgtcaagg gcgctacgga acggttcgtc tcgtcgccgg aggatgtttt cgaggtgatc
      601 gaggagggca aatccaatcg tcacatcgct gtgacaaaca tgaacgagca ttcttcgcga
      661 tcccactcag tattccttat caatgtgaag caggagaacc tggagaacca gaagaaacta
      721 tccggcaaac tctacctggt ggatttggcc ggatccgaga aggtttccaa gactggagcg
      781 gagggaaccg ttcttgatga agccaagaac atcaacaagt cgctgtcggc cttgggcaac
      841 gtaatttctg ccctggcgga cggaaacaaa acgcacatcc cctaccgtga ttccaagcta
      901 acgcgcatcc tgcaggagtc gctgggaggc aacgcacgca caaccatcgt catctgctgc
      961 tctccagcca gtttcaacga atctgaaacg aagtcaacgc tggacttcgg tcgtagagcc
     1021 aagacagtga agaacgtggt ctgcgttaac gaggagctta ctgccgagga atggaagcga
     1081 cgctatgaaa aggagaagga aaagaacgcc cgactaaagg gtaaggtgga gaagcttgag
     1141 atcgagcttg cgcgctggag agcgggtgaa actgttaagg cggaggagca aatcaacatg
     1201 gaggatctca tggaggcaag cacgcccaac ctggaagtgg aggcagcaca gacggcggcg
     1261 gccgaggccg ctttggccgc ccagcgaacg gctctcgcca atatgtccgc atcggttgcc
     1321 gtgaacgagc aggccaggct ggctacagag tgcgagcgtc tctaccagca gctggacgac
     1381 aaggatgagg agatcaatca gcagagccag tacgccgagc agctcaagga gcaggtgatg
     1441 gagcaggagg aactcatcgc taacgctcgg cgtgagtatg agactttgca gtcggagatg
     1501 gcgcgaattc aacaggagaa cgagtccgcc aaggaagagg ttaaggaggt gctccaagct
     1561 ctcgaagagc tgactgtaaa ctacgaccag aaatcccagg agatcgataa caagaacaag
     1621 gatatcgatg ccctcaacga ggagctgcag cagaagcagt ctgtgttcaa cgccgcctcc
     1681 acagagctac agcagctcaa gatgcttgtt ccgcgtggga gcggcggcgg tctgaacgat
     1741 attttcgaag cgcagaaaat tgagtggcat gaacatcatc atcatcatca ctaagcggcc
     1801 gcgctagcaa ttaggctcac attagcgatt aactttaaga aggagatata catatgtccc
     1861 ctatactagg ttattggaaa attaagggcc ttgtgcaacc cactcgactt cttttggaat
     1921 atcttgaaga aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa
     1981 acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc ttattatatt gatggtgatg
     2041 ttaaattaac acagtctatg gccatcatac gttatatagc tgacaagcac aacatgttgg
     2101 gtggttgtcc aaaagagcgt gcagagattt caatgcttga aggagcggtt ttggatatta
     2161 gatacggtgt ttcgagaatt gcatatagta aagactttga aactctcaaa gttgattttc
     2221 ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt
     2281 taaatggtga tcatgtaacc catcctgact tcatgttgta tgacgctctt gatgttgttt
     2341 tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta
     2401 ttgaagctat cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt
     2461 tgcagggctg gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg gatctggttc
     2521 cgcgtggatc caaggataac accgtgccac tgaaattgat tgccctgtta gcgaacggtg
     2581 aatttcactc tggcgagcag ttgggtgaaa cgctgggaat gagccgggcg gctattaata
     2641 aacacattca gacactgcgt gactggggcg ttgatgtctt taccgttccg ggtaaaggat
     2701 acagcctgcc tgagcctatc cagttactta atgctaaaca gatattgggt cagctggatg
     2761 gcggtagtgt agccgtgctg ccagtgattg actccacgaa tcagtacctt cttgatcgta
     2821 tcggagagct taaatcgggc gatgcttgca ttgcagaata ccagcaggct ggccgtggtc
     2881 gccggggtcg gaaatggttt tcgccttttg gcgcaaactt atatttgtcg atgttctggc
     2941 gtctggaaca aggcccggcg gcggcgattg gtttaagtct ggttatcggt atcgtgatgg
     3001 cggaagtatt acgcaagctg ggtgcagata aagttcgtgt taaatggcct aatgacctct
     3061 atctgcagga tcgcaagctg gcaggcattc tggtggagct gactggcaaa actggcgatg
     3121 cggcgcaaat agtcattgga gccgggatca acatggcaat gcgccgtgtt gaagagagtg
     3181 tcgttaatca ggggtggatc acgctgcagg aagcggggat caatctcgat cgtaatacgt
     3241 tggcggccat gctaatacgt gaattacgtg ctgcgttgga actcttcgaa caagaaggat
     3301 tggcacctta tctgtcgcgc tgggaaaagc tggataattt tattaatcgc ccagtgaaac
     3361 ttatcattgg tgataaagaa atatttggca tttcacgcgg aatagacaaa cagggggctt
     3421 tattacttga gcaggatgga ataataaaac cctggatggg cggtgaaata tccctgcgta
     3481 gtgcagaaaa ataagagctc gggcccactc gatcgagcac caccaccacc accactgaga
     3541 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     3601 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     3661 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3721 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3781 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3841 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3901 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3961 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4021 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4081 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     4141 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4201 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4261 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4321 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4381 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4441 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4501 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4561 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4621 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4681 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4741 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4801 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4861 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4921 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4981 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5041 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5101 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5161 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5221 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5281 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5341 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5401 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     5461 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5521 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5581 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5641 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5701 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5761 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5821 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5881 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5941 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6001 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6061 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     6121 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     6181 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     6241 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     6301 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     6361 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     6421 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     6481 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     6541 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     6601 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     6661 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     6721 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     6781 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     6841 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     6901 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     6961 atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa
     7021 tacgactcac tatagggaga ccacaacggt ttccctctag aaataat
//
