LOCUS       sequence-263589-        6080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Drosophila Kinesin-1 with extended neck linker (DAL 
            insert) truncated at 560 and GFP tagged for motility assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6080)
  AUTHORS   Hancock
  TITLE     Hancock Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6080)
  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            "Unpublished"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_17NL
FEATURES             Location/Qualifiers
     source          1..6080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             32..54
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             1582..1590
                     /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)"
     regulatory      1790..1799
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1796..1798,1799..1801,1802..2512)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1796..1798,1799..1801,1802..2512)
                     /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(1841..1862)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2102..2121)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2449..2470
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2522..2539
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2562..2579
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2632..2650)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2646..2693
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2730..3185
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2817..2836)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3027..3048
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3211..3315
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3316..3384,3385..4176)
                     /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(3534..3553)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4347..4935
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4836..4855
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5089..5106
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5121..5260
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5186..5205)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5346..5368
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5362..5553)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     join(6062..6080,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6062..6080
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gagaccacaa cggtttccct ctagaaataa ttttgtttaa ctttaagaag gagatataca
       61 tatgagcgca gaacgagaaa ttccagccga ggacagcatc aaagtggtct gccgattccg
      121 accgctgaac gacagcgaag agaaggccgg ctccaagttc gtggtcaagt tccccaacaa
      181 tgtggaggag aactgcatat ccatagcggg caaggtgtat ttgttcgaca aggtcttcaa
      241 accgaatgca tcccaggaaa aggtctacaa tgaggcggcc aagtccattg ttacggatgt
      301 cctggccggg tacaatggaa cgatattcgc atacggtcag acgtcctccg gaaaaacgca
      361 tacgatggag ggcgtgatcg gggactccgt aaaacagggt atcataccta ggatcgtcaa
      421 cgacattttc aatcacatct acgcgatgga ggtgaacctg gagtttcaca tcaaggtctc
      481 ctactacgag atctacatgg acaagattcg agatctgttg gacgtctcca aggtgaacct
      541 tagtgtgcac gaggataaga accgggtgcc gtacgtcaag ggcgctacgg aacggttcgt
      601 ctcgtcgccg gaggatgttt tcgaggtgat cgaggagggc aaatccaatc gtcacatcgc
      661 tgtgacaaac atgaacgagc attcttcgcg atcccactca gtattcctta tcaatgtgaa
      721 gcaggagaac ctggagaacc agaagaaact atccggcaaa ctctacctgg tggatttggc
      781 cggatccgag aaggtttcca agactggagc ggagggaacc gttcttgatg aagccaagaa
      841 catcaacaag tcgctgtcgg ccttgggcaa cgtaatttct gccctggcgg acggaaacaa
      901 aacgcacatc ccctaccgtg attccaagct aacgcgcatc ctgcaggagt cgctgggagg
      961 caacgcacgc acaaccatcg tcatctgctg ctctccagcc agtttcaacg aatctgaaac
     1021 gaagtcaacg ctggacttcg gtcgtagagc caagacagtg aagaacgtgg tctgcgttaa
     1081 cgaggagctt actgatgctc tagccgagga atggaagcga cgctatgaaa aggagaagga
     1141 aaagaacgcc cgactaaagg gtaaggtgga gaagcttgag atcgagcttg cgcgctggag
     1201 agcgggtgaa actgttaagg cggaggagca aatcaacatg gaggatctca tggaggcaag
     1261 cacgcccaac ctggaagtgg aggcagcaca gacggcggcg gccgaggccg ctttggccgc
     1321 ccagcgaacg gctctcgcca atatgtccgc atcggttgcc gtgaacgagc aggccaggct
     1381 ggctacagag tgcgagcgtc tctaccagca gctggacgac aaggatgagg agatcaatca
     1441 gcagagccag tacgccgagc agctcaagga gcaggtgatg gagcaggagg aactcatcgc
     1501 taacgctcgg cgtgagtatg agactttgca gtcggagatg gcgcgaattc aacaggagaa
     1561 cgagtccgcc aaggaagagg ttaaggaggt gctccaagct ctcgaagagc tgactgtaaa
     1621 ctacgaccag aaatcccagg agatcgataa caagaacaag gatatcgatg ccctcaacga
     1681 ggagctgcag cagaagcagt ctgtgttcaa cgccgcctcc acagagctac agcagctcaa
     1741 gatgcttgtt ccgcgtgggc ccgtgcaccg cctaggggat ccaccggtcg ccaccatggt
     1801 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1861 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1921 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1981 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     2041 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2101 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2161 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2221 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     2281 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     2341 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2401 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2461 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agctcgagag
     2521 ccatcatcat catcatcact aagcggccgc actcgatcga gcaccaccac caccaccact
     2581 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     2641 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     2701 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     2761 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2821 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2881 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2941 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3001 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3061 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3121 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     3181 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3241 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3301 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3361 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3421 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3481 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3541 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3601 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3661 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3721 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3781 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3841 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3901 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3961 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4021 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4081 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4141 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4201 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4261 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4321 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4381 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4441 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4501 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4561 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4621 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4681 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4741 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4801 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4861 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4921 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4981 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5041 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     5101 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5161 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     5221 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     5281 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5341 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     5401 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     5461 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     5521 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     5581 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     5641 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     5701 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     5761 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     5821 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     5881 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     5941 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     6001 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatctcga tcccgcgaaa
     6061 ttaatacgac tcactatagg
//
