LOCUS       sequence-460709-        6136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kif3A (aa1-342)-Kif1A neck linker(17aa) fused to Kin1 coiled coil 
            (aa345-406)-EGFP-SNAP-his. This Kif3A was used to link an oligo to 
            the motor using the SNAP tag for single molecule experiments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6136)
  TITLE     DNA tensiometer reveals catch-bond detachment kinetics of kinesin-1,
            -2 and -3
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6136)
  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            "Unpublished"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: Kif3A-EGFP-SNAP
FEATURES             Location/Qualifiers
     source          1..6136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        493..597
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(598..666,667..1458)
                     /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(816..835)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1629..2217
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2118..2137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2371..2388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2403..2542
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2468..2487)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2628..2650
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2644..2835)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3344..3363
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3344..3362
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             3394..3416
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      4687..4696
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4693..4695,4696..4698,4699..5409)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4693..4695,4696..4698,4699..5409)
                     /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(4738..4759)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4999..5018)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5346..5367
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5428..5973
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             5980..5997
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6050..6068)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6064..6111
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
      601 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
      661 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
      721 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
      781 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      841 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      901 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      961 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1021 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1081 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1141 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1201 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     1261 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     1321 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     1381 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     1441 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1501 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     1561 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1621 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1681 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1741 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     1801 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1861 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1921 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1981 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2041 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2101 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2161 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2221 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     2281 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     2341 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     2401 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     2461 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     2521 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     2581 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2641 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     2701 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     2761 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     2821 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     2881 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     2941 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     3001 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     3061 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     3121 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     3181 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     3241 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     3301 tggccaggac ccaacgctgc ccgagatctc gatcccgcga aattaatacg actcactata
     3361 gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga aggagatata
     3421 catatgccga tcaataagtc ggagaagccg gaaagctgcg ataatgtgaa ggtggtggtt
     3481 aggtgccggc ctctcaatga gagagagaag tcaatgtgct atagacaggc cgtcagcgtg
     3541 gatgaaatga ggggaactat caccgtccat aagaccgatt cctccaacga acctccaaag
     3601 acatttactt tcgatactgt ttttggacca gaaagtaaac aacttgatgt gtataactta
     3661 actgcaagac caattattga ttctgttctg gaaggctaca atgggaccat ctttgcatat
     3721 ggacagaccg gaacaggcaa gactttcacc atggaaggcg ttcgagcagt accggggctc
     3781 agaggggtca tccccaactc atttgctcac atatttggtc acatcgcaaa agcagagggc
     3841 gacacaaggt ttttggttcg agtgtcatac ttggagatat ataatgaaga agttcgagac
     3901 cttttgggta aagatcagac ccagaggtta gaggttaaag aaaggcctga tgtgggagta
     3961 tatatcaaag acttatcagc atatgtagtc aacaacgctg acgacatgga tagaatcatg
     4021 actctaggcc acaaaaaccg ttctgtcggt gcgactaata tgaacgagca cagctcccga
     4081 tcccacgcca tctttacaat tactatagag tgcagtgaga aaggcgtgga cgggaacatg
     4141 cacgtcagga tgggcaagct ccaccttgtg gatctggctg ggtcagaaag acaagcaaaa
     4201 actggagcca caggacagcg cctcaaggaa gctaccaaaa tcaacctttc attgtctacc
     4261 ctgggcaacg ttatttctgc cttggtggat ggaaaaagca ctcacgtgcc gtatcggaac
     4321 tccaaactga cccgtctcct gcaggactcc ttaggaggga actcaaaaac catgatgtgt
     4381 gcaaatattg ggccagcaga ttataattat gatgaaacta tcagcacctt gcggtatgcc
     4441 aaccgcgcca agaacattaa aaacaaagcg agaatcaatg aggacccaaa ggatgcgcta
     4501 gccgaggaat ggaagcgacg ctatgaaaag gagaaggaaa agaacgcccg actaaagggt
     4561 aaggtggaga agcttgagat cgagcttgcg cgctggagag cgggtgaaac tgttaaggcg
     4621 gaggagcaaa tcaacatgga ggatctcatg gaggcaagca cgcccaacct ggaagtggag
     4681 gcagcagcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     4741 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     4801 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     4861 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     4921 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     4981 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     5041 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     5101 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     5161 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     5221 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     5281 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     5341 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     5401 ctgtacaagc ttaccggtga attcgctatg gacaaagatt gcgaaatgaa acgtaccacc
     5461 ctggatagcc cgctgggcaa actggaactg agcggctgcg aacagggcct gcatgaaatt
     5521 aaactgctgg gtaaaggcac cagcgcggcc gatgcggttg aagttccggc cccggccgcc
     5581 gtgctgggtg gtccggaacc gctgatgcag gcgaccgcgt ggctgaacgc gtattttcat
     5641 cagccggaag cgattgaaga atttccggtt ccggcgctgc atcatccggt gtttcagcag
     5701 gagagcttta cccgtcaggt gctgtggaaa ctgctgaaag tggttaaatt tggcgaagtg
     5761 attagctatc agcagctggc ggccctggcg ggtaatccgg cggccaccgc cgccgttaaa
     5821 accgcgctga gcggtaaccc ggtgccgatt ctgattccgt gccatcgtgt ggttagctct
     5881 agcggtgcgg ttggcggtta tgaaggtggt ctggcggtga aagagtggct gctggcccat
     5941 gaaggtcatc gtctgggtaa accgggtctg ggacctgcac accaccacca ccaccactga
     6001 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     6061 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     6121 ggaactatat ccggat
//
