LOCUS       sequence-422377-        6071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Drosophila Kinesin-1 truncated after coil 1 at 560 and 
            tagged with GFP for single-molecule motor assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6071)
  AUTHORS   Hancock
  TITLE     Hancock Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6071)
  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            "Unpublished"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: K560GFP
FEATURES             Location/Qualifiers
     source          1..6071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             88..110
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(141..160)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(142..160)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             669..860
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(854..876)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    962..1101
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1017..1036
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1116..1133)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1287..1875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1367..1386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2046..2837,2838..2906))
                     /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     2669..2688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2907..3011)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3037..3492)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3174..3195)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3386..3405
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3529..3576)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3572..3590
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3643..3660)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3683..3700)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(3710..4420,4421..4423,4424..4426))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(3710..4420,4421..4423,4424..4426))
                     /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(3752..3773)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4101..4120
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4360..4381
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4423..4432
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             4632..4640
                     /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)"
ORIGIN
        1 tcttcgctgt cgttcagcgg tcggaatcgg cagaccactt tgatgctgtc ctcggctgga
       61 atttctcgtt ctgcgctcat atgtatatct ccttcttaaa gttaaacaaa attatttcta
      121 gagggaaacc gttgtggtct ccctatagtg agtcgtatta atttcgcggg atcgagatct
      181 cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc
      241 ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa
      301 cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg
      361 gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc
      421 ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga
      481 agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt
      541 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga
      601 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg
      661 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc
      721 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct
      781 gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg
      841 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
      901 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
      961 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     1021 atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta
     1081 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     1141 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     1201 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1261 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1321 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1381 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1441 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1501 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1561 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1621 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1681 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     1741 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     1801 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     1861 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     1921 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     1981 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2041 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     2101 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     2161 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     2221 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     2281 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     2341 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     2401 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     2461 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     2521 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     2581 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     2641 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     2701 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     2761 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     2821 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     2881 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     2941 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3001 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     3061 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     3121 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     3181 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     3241 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     3301 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     3361 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     3421 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     3481 tacagggcgc gtcccattcg ccaatccgga tatagttcct cctttcagca aaaaacccct
     3541 caagacccgt ttagaggccc caaggggtta tgctagttat tgctcagcgg tggcagcagc
     3601 caactcagct tcctttcggg ctttgttagc agccggatct cagtggtggt ggtggtggtg
     3661 ctcgatcgag tgcggccgct tagtgatgat gatgatgatg gctctcgagc ttgtacagct
     3721 cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc
     3781 gcttctcgtt ggggtctttg ctcagggcgg actgggtgct caggtagtgg ttgtcgggca
     3841 gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg agctgcacgc
     3901 tgccgtcctc gatgttgtgg cggatcttga agttcacctt gatgccgttc ttctgcttgt
     3961 cggccatgat atagacgttg tggctgttgt agttgtactc cagcttgtgc cccaggatgt
     4021 tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg gtgtcgccct
     4081 cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct
     4141 cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc atgtggtcgg
     4201 ggtagcggct gaagcactgc acgccgtagg tcagggtggt cacgagggtg ggccagggca
     4261 cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag gtggcatcgc
     4321 cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca
     4381 ggatgggcac caccccggtg aacagctcct cgcccttgct caccatggtg gcgaccggtg
     4441 gatcccctag gcggtgcacg ggcccacgcg gaacaagcat cttgagctgc tgtagctctg
     4501 tggaggcggc gttgaacaca gactgcttct gctgcagctc ctcgttgagg gcatcgatat
     4561 ccttgttctt gttatcgatc tcctgggatt tctggtcgta gtttacagtc agctcttcga
     4621 gagcttggag cacctcctta acctcttcct tggcggactc gttctcctgt tgaattcgcg
     4681 ccatctccga ctgcaaagtc tcatactcac gccgagcgtt agcgatgagt tcctcctgct
     4741 ccatcacctg ctccttgagc tgctcggcgt actggctctg ctgattgatc tcctcatcct
     4801 tgtcgtccag ctgctggtag agacgctcgc actctgtagc cagcctggcc tgctcgttca
     4861 cggcaaccga tgcggacata ttggcgagag ccgttcgctg ggcggccaaa gcggcctcgg
     4921 ccgccgccgt ctgtgctgcc tccacttcca ggttgggcgt gcttgcctcc atgagatcct
     4981 ccatgttgat ttgctcctcc gccttaacag tttcacccgc tctccagcgc gcaagctcga
     5041 tctcaagctt ctccacctta ccctttagtc gggcgttctt ttccttctcc ttttcatagc
     5101 gtcgcttcca ttcctcggca gtaagctcct cgttaacgca gaccacgttc ttcactgtct
     5161 tggctctacg accgaagtcc agcgttgact tcgtttcaga ttcgttgaaa ctggctggag
     5221 agcagcagat gacgatggtt gtgcgtgcgt tgcctcccag cgactcctgc aggatgcgcg
     5281 ttagcttgga atcacggtag gggatgtgcg ttttgtttcc gtccgccagg gcagaaatta
     5341 cgttgcccaa ggccgacagc gacttgttga tgttcttggc ttcatcaaga acggttccct
     5401 ccgctccagt cttggaaacc ttctcggatc cggccaaatc caccaggtag agtttgccgg
     5461 atagtttctt ctggttctcc aggttctcct gcttcacatt gataaggaat actgagtggg
     5521 atcgcgaaga atgctcgttc atgtttgtca cagcgatgtg acgattggat ttgccctcct
     5581 cgatcacctc gaaaacatcc tccggcgacg agacgaaccg ttccgtagcg cccttgacgt
     5641 acggcacccg gttcttatcc tcgtgcacac taaggttcac cttggagacg tccaacagat
     5701 ctcgaatctt gtccatgtag atctcgtagt aggagacctt gatgtgaaac tccaggttca
     5761 cctccatcgc gtagatgtga ttgaaaatgt cgttgacgat cctaggtatg ataccctgtt
     5821 ttacggagtc cccgatcacg ccctccatcg tatgcgtttt tccggaggac gtctgaccgt
     5881 atgcgaatat cgttccattg tacccggcca ggacatccgt aacaatggac ttggccgcct
     5941 cattgtagac cttttcctgg gatgcattcg gtttgaagac cttgtcgaac aaatacacct
     6001 tgcccgctat ggatatgcag ttctcctcca cattgttggg gaacttgacc acgaacttgg
     6061 agccggcctt c
//
