LOCUS       sequence-435682-        7859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of KIF5B(1-560)-HaloTag-Strep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7859)
  AUTHORS   Smith ER, Turner ED, Abdelhamid MAS, Craggs TD, Twelvetrees AE
  TITLE     Kinesin-1 is highly flexible and adopts an open conformation in the 
            absence of cargo
  JOURNAL   bioRxiv 2024.12.20.629623
REFERENCE   2  (bases 1 to 7859)
  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: "bioRxiv 
            2024.12.20.629623"
COMMENT     Sequence Label: K560-Halo-Strep
FEATURES             Location/Qualifiers
     source          1..7859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1520..1542
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(1554..1573)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1555..1573)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1615..1634)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          1617..1749
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        complement(1885..2088)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1914..1934)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2089..2468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    2659..2942
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     rep_origin      3058..3646
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3547..3566
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3804..4595,4596..4664))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4427..4446
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4665..4769)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4925..4973
                     /note="synthetic polyadenylation signal"
     CDS             complement(5037..5831)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5148..5167)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5758..5777
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5882..6239)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5896..6031
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6048..6239
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6196..6216
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    6484..6605
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6521..6540)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6575..6594
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6642..6660
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6732..6755)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(6756..7640)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             complement(7656..7676)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
ORIGIN
        1 ctgatcataa ttgacagcaa gttcttctag ggcctgtaaa acttctttca cttcttcttt
       61 agaggcatca ttttctgctt gaaggcgatt cagctcagct tgcatattgt cttgatccct
      121 tctggtagat gccaaaagct cctcctgatc caacatttgc gtcttcagct tctctaccag
      181 ttgactttgc tggttaattt cttcatcctt gtcatcaagc tgtttgtata atttagcaat
      241 ttcttcttca cactttcttc tttcagcatc agtaaaattt cctataactc caattgcggt
      301 tgctggttta tcattggtaa gagtaatatc tttatccact gtgaaagctt ccaagttggc
      361 tttctctttg tcaaactgtt catcaatagg caccgtctcc ccattacgcc atctgttgag
      421 ctcattttca agccactgaa tagtgttccg caggatctta tttttttctt tttctttttc
      481 atacttcttt ttccactgtt ctgcagttaa ctccacattg acacaaactg tgttcttaat
      541 tgttttggcc ctttggccaa ataagagtgt agattttgtt tcagactcat tgtatgatga
      601 tggagagcag caaattacaa tagtggttct acagttgcca cctaatgaat cttgaaggat
      661 tcttgtcatt ttactatctc gatatggaac ataagtacta ccctcagcca aagcagaaat
      721 aacatttcca agagcagaaa gtgacttgtt gatgttttta gcttcatcca gcacagcacc
      781 ttcagctcca gttttactaa ccttttcact accagctaaa tcaactagat aaagttttcc
      841 actcagcttt tgttccgttt gtgtgttctc ttgtttgaca ttaataagaa atatactgtg
      901 actcctagag ctatgttcat tcatatttgt aactgctaca tgtctgttgg attttccttc
      961 atctatggta tccataactt catctggact acatacaaaa cgctctgtgc acccctttac
     1021 atagggaact cggtttttgt cttcatgaac tgaaaggttg gtctttgaaa catctaacag
     1081 gtcccttatc ttatccaaat atatttcaaa atatgaaacc ttaatatgaa attccaaatt
     1141 ttcatccatg gagtaaatat aattaaaaat atcttgcact attcttggaa taattcccat
     1201 gccttctgga tcatgaagtt taccctccat tgtgtgtgtc ttcccagagg atgtttggcc
     1261 gtatgcaaat attgttccat tatatccttc aagtacatct ttaacaatct tctttgcaca
     1321 gtcattatac acttgctctt gagatgtgct tgactggaac acccgatcaa atgcataagg
     1381 cttggacgcg atcacgaccg tgtcttctcc ctgaaacttg gcgatgtact tgtcgccgcg
     1441 gttcacttca gactcgttga gaggtctgaa gcgacacatc actttgatgt tgcactcggc
     1501 caggtccgcc atatgtatat ctccttctta aagttaaaca aaattatttc tagccctata
     1561 gtgagtcgta ttaatactct agccttaaga gctgtaattg aactgggagt ggacacctgt
     1621 ggagagaaag gcaaagtgga tgtcagtaag accaataggt gcctatcaga aacgcaagag
     1681 tcttctctgt ctcgacaagc ccagtttcta ttggtctcct taaacctgtc ttgtaacctt
     1741 gatacttacc tgcccagtgc ctcacgacca acttctgcag cttaagttcg agactgttgt
     1801 gtcagaagca ctgactgcgt tagcaattta actgtgataa actaccgcaa taaagcttct
     1861 agtgatctga cggttcacta aaccagctct gcttatatag acctcccacc gtacacgcct
     1921 accgcccatt tgcgtcaacg gggcggggtt attacgacat tttggaaagt cccgttgatt
     1981 ttggtgccaa aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga
     2041 gtcaaaccgc tatccacgcc cattggtgta ctgccaaaac cgcatcacca tggtaatagc
     2101 gatgactaat acgtagatgt actgccaagt aggaaagtcc cgtaaggtca tgtactgggc
     2161 ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcggac ttggcatatg
     2221 atacacttga tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca
     2281 atggaaagtc cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg
     2341 ggggtcgttg ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc
     2401 catatatggg ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa
     2461 tcaatgccaa catggcggtc atattggaca tgagccaata taaatgtaca tattatgata
     2521 tagatacaac gtatgcaatg gccaatagcc aatattgatt tatgctatat aaccaatgaa
     2581 taatatggct aatggccaat attgaagatc tcgatcctct acgccggacg catcgtggcc
     2641 ggagtactgc ggtttatctc gatggctacg agggcagaca gtaagtggat ttaccataat
     2701 cccttaattg tacgcaccgc taaaacgcgt tcagcgcgat cacggcagca gacaggtaaa
     2761 aatggcaaca aaccacccga aaaactgccg caatcgcgcc tgataaattt taaccgtatg
     2821 aatacctatg caaccagagg gtacaggcca cattaccccc acttaatcca ctgaagctgc
     2881 catttttcat ggtttcacca tcccagcgaa gggccatcca gcgtgcgttc ctgtatttcc
     2941 ggactataaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3001 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3061 aaatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3121 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3181 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     3241 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3301 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3361 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3421 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3481 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3541 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3601 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3661 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3721 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccggttgggc
     3781 gtcgcttggt cggtcatttc gaattaccaa tgcttaatca gtgaggcacc tatctcagcg
     3841 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3901 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3961 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4021 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4081 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4141 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4201 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatagt tgtcagaagt
     4261 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4321 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4381 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4441 catagcagaa ctttgaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4501 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4561 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4621 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4681 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4741 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4801 taagaaacca ttacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
     4861 agctactggg ctatctggac aagggaaaac gcaagcgcaa agaggatcct tatcgctatc
     4921 gattcacaca aaaaaccaac acacagatgt aatgaaaata aagatatttt attgcggcca
     4981 tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg aaccccagag tcccgctcag
     5041 aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg
     5101 taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta
     5161 gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca
     5221 gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg
     5281 agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc
     5341 ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt
     5401 gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta
     5461 tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat
     5521 gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg
     5581 acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct
     5641 gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg
     5701 cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc
     5761 cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct
     5821 tgttcaatca tggtggctct agccttaagt tcgagactgt tgtgtcagaa gaatcaagct
     5881 ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg
     5941 ccgaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat
     6001 gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg
     6061 ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc
     6121 cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     6181 ctggggactt tccacaccct aactgacaca cattccacag ctggttcttt ccgcctcaga
     6241 aggtacctaa ccaagttcct ctttcagagg ttatttcagg ccatggtgct gcgcagatcc
     6301 gcgtatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt
     6361 gtaagcgtta atattttgtt aaaattcgat tttaccacat ttgtagaggt tttacttgct
     6421 taaaaaaacc tcccacatct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     6481 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     6541 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     6601 tcttatcatg tctgctcgaa gcggccgccc caaggggtta tgctagttat tgctcagcgg
     6661 tggcagcagc caactcagct tcctttcggg ctttgttagc agccggatca gcttgcatgc
     6721 caattctatt atttttcaaa ctgcggatgg ctccaaccgg aaatctccag agtagacagc
     6781 cagcgcgcga tctcgctgcc gatcaggtcc gggttgtctt cttgcagcag attcagaccc
     6841 gggccgatgt ccacagcctt gcagttaggc aggcttttgg ccaggcgagc ggcttcggcc
     6901 ggtgggatca gaacgcctgg ggtgccccag aacagcagct tcgggacagg ggactggtgc
     6961 agccagtcca tgtattcttc gaccagcgcg acgatgttcg ctggctcacc ggcgattggc
     7021 agctcgtttg ggaagcgcca cagtggctcg cggtcaacag gattcaggaa cggctcgcgg
     7081 taatggtcca tctcgacttc agtcagcggg cggacgacac ccatcggcag cgtaccctcg
     7141 ataaaaacgt tctgatcgat gatcagcttg cggccgacgt cggtggtgcg gaaggcctgg
     7201 aaggtctcgc gggcaaattc tggccattcg tcccaggtcg ggatagggcg gatgaactcc
     7261 ataaatgcaa tacctttgac gcgctctgga ttgcgcttgg cccagtggaa acccagagcg
     7321 gagccccagt cgtgaatgac caggacgacc tcttccagac ccagggcttc gatgaaggca
     7381 tccatgaagc ggacgtggtc gtcgaagaaa taacccaggt ctggtttgtc ggatttgccc
     7441 ataccgatca ggtctggagc aatgcagcga tgggtcggtg caacatgcgg gatgatgttg
     7501 cgccacacgt aggaggaggt cgggttaccg tgcaggaaca gcacaggggt gccatcgcgc
     7561 ggaccaacat cgacgtagtg catgcgctcg cccaggactt ccacataatg ggggtcgaat
     7621 ggaaagccag taccgatttc ggatccatcg ttatcgctct gaaagtacag atcctcagtg
     7681 gttggctcga cttttagtaa agatgccatc atctcagctg ctcgtttttt ctggtggttg
     7741 gtcatttcct taagtttctg aagctcagca tctatactcg ctaaagttgc cgatttctga
     7801 ttcaattcat cactaagcaa ttcatattcc ttagttttgt cttcaacttc ctgagactt
//
