LOCUS       sequence-263552-        7067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Drosophila Kinesin-1 truncated at 560 and biotinylated on 
            the head 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  JOURNAL   Unpublished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            "Unpublished"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_AviN_coBirA
FEATURES             Location/Qualifiers
     source          1..7067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             84..128
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     RBS             1643..1651
                     /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             1806..1823
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1824..1841
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1904..2557
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2515..2537
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2564..2581
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2582..3544
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2582..3541
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             3568..3585
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3638..3656)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3652..3699
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3736..4191
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3823..3842)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4033..4054
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4217..4321
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4322..4390,4391..5182)
                     /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(4540..4559)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5353..5941
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5842..5861
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6095..6112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6127..6266
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6192..6211)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6352..6374
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6368..6559)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atgggtctga acgatatttt cgaagcgcag aaaattgagt
      121 ggcatgaagg cggcagcgca gaacgagaaa ttccagccga ggacagcatc aaagtggtct
      181 gccgattccg accgctgaac gacagcgaag agaaggccgg ctccaagttc gtggtcaagt
      241 tccccaacaa tgtggaggag aactgcatat ccatagcggg caaggtgtat ttgttcgaca
      301 aggtcttcaa accgaatgca tcccaggaaa aggtctacaa tgaggcggcc aagtccattg
      361 ttacggatgt cctggccggg tacaatggaa cgatattcgc atacggtcag acgtcctccg
      421 gaaaaacgca tacgatggag ggcgtgatcg gggactccgt aaaacagggt atcataccta
      481 ggatcgtcaa cgacattttc aatcacatct acgcgatgga ggtgaacctg gagtttcaca
      541 tcaaggtctc ctactacgag atctacatgg acaagattcg agatctgttg gacgtctcca
      601 aggtgaacct tagtgtgcac gaggataaga accgggtgcc gtacgtcaag ggcgctacgg
      661 aacggttcgt ctcgtcgccg gaggatgttt tcgaggtgat cgaggagggc aaatccaatc
      721 gtcacatcgc tgtgacaaac atgaacgagc attcttcgcg atcccactca gtattcctta
      781 tcaatgtgaa gcaggagaac ctggagaacc agaagaaact atccggcaaa ctctacctgg
      841 tggatttggc cggatccgag aaggtttcca agactggagc ggagggaacc gttcttgatg
      901 aagccaagaa catcaacaag tcgctgtcgg ccttgggcaa cgtaatttct gccctggcgg
      961 acggaaacaa aacgcacatc ccctaccgtg attccaagct aacgcgcatc ctgcaggagt
     1021 cgctgggagg caacgcacgc acaaccatcg tcatctgctg ctctccagcc agtttcaacg
     1081 aatctgaaac gaagtcaacg ctggacttcg gtcgtagagc caagacagtg aagaacgtgg
     1141 tctgcgttaa cgaggagctt actgccgagg aatggaagcg acgctatgaa aaggagaagg
     1201 aaaagaacgc ccgactaaag ggtaaggtgg agaagcttga gatcgagctt gcgcgctgga
     1261 gagcgggtga aactgttaag gcggaggagc aaatcaacat ggaggatctc atggaggcaa
     1321 gcacgcccaa cctggaagtg gaggcagcac agacggcggc ggccgaggcc gctttggccg
     1381 cccagcgaac ggctctcgcc aatatgtccg catcggttgc cgtgaacgag caggccaggc
     1441 tggctacaga gtgcgagcgt ctctaccagc agctggacga caaggatgag gagatcaatc
     1501 agcagagcca gtacgccgag cagctcaagg agcaggtgat ggagcaggag gaactcatcg
     1561 ctaacgctcg gcgtgagtat gagactttgc agtcggagat ggcgcgaatt caacaggaga
     1621 acgagtccgc caaggaagag gttaaggagg tgctccaagc tctcgaagag ctgactgtaa
     1681 actacgacca gaaatcccag gagatcgata acaagaacaa ggatatcgat gccctcaacg
     1741 aggagctgca gcagaagcag tctgtgttca acgccgcctc cacagagcta cagcagctca
     1801 agatgcttgt tccgcgtggg agccatcatc atcatcatca ctaagcggcc gcgctagcaa
     1861 ttaggctcac attagcgatt aactttaaga aggagatata catatgtccc ctatactagg
     1921 ttattggaaa attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga
     1981 aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt
     2041 tgaattgggt ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac
     2101 acagtctatg gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc
     2161 aaaagagcgt gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt
     2221 ttcgagaatt gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct
     2281 acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga
     2341 tcatgtaacc catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga
     2401 cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat
     2461 cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg
     2521 gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg gatctggttc cgcgtggatc
     2581 caaggataac accgtgccac tgaaattgat tgccctgtta gcgaacggtg aatttcactc
     2641 tggcgagcag ttgggtgaaa cgctgggaat gagccgggcg gctattaata aacacattca
     2701 gacactgcgt gactggggcg ttgatgtctt taccgttccg ggtaaaggat acagcctgcc
     2761 tgagcctatc cagttactta atgctaaaca gatattgggt cagctggatg gcggtagtgt
     2821 agccgtgctg ccagtgattg actccacgaa tcagtacctt cttgatcgta tcggagagct
     2881 taaatcgggc gatgcttgca ttgcagaata ccagcaggct ggccgtggtc gccggggtcg
     2941 gaaatggttt tcgccttttg gcgcaaactt atatttgtcg atgttctggc gtctggaaca
     3001 aggcccggcg gcggcgattg gtttaagtct ggttatcggt atcgtgatgg cggaagtatt
     3061 acgcaagctg ggtgcagata aagttcgtgt taaatggcct aatgacctct atctgcagga
     3121 tcgcaagctg gcaggcattc tggtggagct gactggcaaa actggcgatg cggcgcaaat
     3181 agtcattgga gccgggatca acatggcaat gcgccgtgtt gaagagagtg tcgttaatca
     3241 ggggtggatc acgctgcagg aagcggggat caatctcgat cgtaatacgt tggcggccat
     3301 gctaatacgt gaattacgtg ctgcgttgga actcttcgaa caagaaggat tggcacctta
     3361 tctgtcgcgc tgggaaaagc tggataattt tattaatcgc ccagtgaaac ttatcattgg
     3421 tgataaagaa atatttggca tttcacgcgg aatagacaaa cagggggctt tattacttga
     3481 gcaggatgga ataataaaac cctggatggg cggtgaaata tccctgcgta gtgcagaaaa
     3541 ataagagctc gggcccactc gatcgagcac caccaccacc accactgaga tccggctgct
     3601 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     3661 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     3721 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3781 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3841 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3901 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3961 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4021 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4081 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4141 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     4201 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4261 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4321 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4381 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4441 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4501 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4561 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4621 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4681 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4741 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4801 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4861 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4921 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4981 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5041 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5101 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5161 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5221 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5281 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5341 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5401 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5461 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     5521 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5581 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5641 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5701 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5761 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5821 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5881 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5941 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6001 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6061 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6121 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     6181 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     6241 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     6301 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     6361 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     6421 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     6481 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     6541 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     6601 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     6661 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     6721 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     6781 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     6841 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     6901 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     6961 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     7021 cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaat
//
