LOCUS       sequence-435690-        9080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of KIF5A-SNAPf-HisTag-FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9080)
  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 9080)
  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: KIF5A-SNAPf-HisTag-FLAG
FEATURES             Location/Qualifiers
     source          1..9080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        175..378
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     329..349
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     423..442
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        423..441
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4142..4159
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4160..4183
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4231..4248)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4237..4461
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4507..4935
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4594..4613)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4804..4825
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4944..4964)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4949..5278
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5129..5264
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5191..5210
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5345..6139
                     /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
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGKVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5399..5418)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6009..6028
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6313..6434
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6350..6369)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6404..6423
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6483..6499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6483..6499)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6496..6518)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6507..6523
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6531..6537,6538..6555,6556..6561))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6576..6597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6714..6731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6885..7473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6965..6984)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7644..8435,8436..8504))
                     /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     8267..8286
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8505..8609)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8684..8703)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8875..9080,1..174)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
       61 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      121 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      181 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      241 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      301 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      361 gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa
      421 attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt aagctatggc
      481 ggagactaac aacgaatgca gcatcaaggt gctttgccga tttcggcccc tgaaccaggc
      541 cgagattctg cggggggaca agttcatccc cattttccaa ggggacgaca gcgtcattat
      601 tgggggaaag ccatatgtct ttgaccgcgt cttcccccca aacaccactc aggagcaggt
      661 ttaccacgcc tgtgccatgc agatcgtcaa agacgtcctt gctggttaca atggcacaat
      721 cttcgcttat ggacagacat cctcagggaa aacgcatacc atggagggga agctgcacga
      781 ccctcagctg atgggcatca ttccccggat cgctcgagac atcttcaacc acatctactc
      841 catggatgag aaccttgaat tccacattaa ggtatcttac ttcgagattt acctggataa
      901 gatccgtgac cttttggatg tgaccaagac gaacctgtcc gtgcatgagg acaaaaaccg
      961 ggtgccgttt gtcaagggtt gtaccgaacg ctttgtgtcc agcccagagg agattctgga
     1021 tgtgatcgat gaggggaagt ccaaccgtca cgtagctgtc accaacatga acgagcacag
     1081 ttctcggagc cacagcatct tcctcatcaa catcaagcag gagaacgtag agaccgagca
     1141 gaagctcagc gggaagctgt acctcgtgga tctggccgga agcgagaagg tcagcaagac
     1201 aggggcagag ggagccgttc tggacgaggc aaagaatatc aacaagtcgc tgtcggccct
     1261 ggggaacgtg atctctgcac tggcagaggg caccaaaagc tacgtgccgt accgcgacag
     1321 caaaatgacg aggattctcc aggactctct gggagggaac tgcaggacta ccatgttcat
     1381 ctgctgctcg ccgtccagct acaatgacgc agagaccaag tccacgctca tgtttggaca
     1441 gcgggcgaag accatcaaga acactgcctc agtgaatctg gagctgactg ctgagcagtg
     1501 gaagaagaag tatgagaagg agaaggagaa gaccaaggcc cagaaggaga caattgcgaa
     1561 gctagaggct gagcttagcc ggtggcgcaa tggagagaat gtgcctgaga ctgagcgcct
     1621 ggctggagag gactcagctc tgggagctga gctctgcgag gagacccctg tgaatgacaa
     1681 ctcatccatt gtggtacgca tcgcacctga ggaaaggcag aaatatgagg aagagatccg
     1741 ccgtctctac aagcagcttg atgacaagga tgatgagatc aaccagcaga gccagctcat
     1801 tgagaagctg aagcagcaga tgctggacca ggaagagctg ctcgtgtcca ctcggggaga
     1861 caacgagaag gtccagcggg agcttagcca cctgcagtcc gagaacgatg ctgcgaagga
     1921 cgaggtgaag gaagtgctgc aggccctaga ggagctggcg gtcaactacg accagaagtc
     1981 ccaggaggtg gaggagaaga gccagcagaa ccagctgctg gtggacgagc tgtcccagaa
     2041 agtggccacc atgctgtccc tggagtccga gctacagcgg ctccaggagg tcagtggaca
     2101 ccagcgaaag cggatcgctg aggtgctgaa tgggctgatg agggacctga gtgagttcag
     2161 tgtcatcgtg ggcaacggcg agattaagct gcccgtggag atcagtgggg ccatcgagga
     2221 ggagttcacg gtggcccggc tctacatcag caagatcaag tcggaggtga agtccgtggt
     2281 taagcgatgt cggcagctgg agaacctcca ggtggagtgt catcgcaaga tggaggtgac
     2341 cggtagggag ctgtcatctt gccaactgct catctcacag catgaggcca agatccgttc
     2401 actcacggag tacatgcaga ctgtggagtt gaagaaacgg cacctggaag agtcctacga
     2461 ctccctgagc gatgagcttg ccaggctcca ggcgcacgaa actgtacacg aggtagctct
     2521 gaaagacaag gagccagaca cacaggacgc ggaggaggtg aagaaggccc tggaactaca
     2581 gatggagaat caccgtgagg cccatcaccg gcagctggcc cgcctccggg atgagattaa
     2641 tgagaaacag aaaaccattg atgagctgaa agacctgaac cagaagctcc agttagagct
     2701 ggagaagctt caggccgact atgagaggct gaagaatgaa gagaacgaga agagcgccaa
     2761 gctccaggag ctgacatttc tgtatgagcg acatgagcag tccaagcagg acctcaaggg
     2821 gctggaggag acagttgccc gtgaactcca gaccctccac aaccttcgca agctgttcgt
     2881 tcaagacgtc acgactcgag tcaagaaaag tgcagaaatg gagcccgagg acagtggggg
     2941 gattcattcc caaaagcaga agatctcctt tcttgagaac aacctggaac agcttacaaa
     3001 ggttcacaaa cagctggtac gtgacaatgc agatctgcgt tgtgagcttc ctaaattgga
     3061 aaaacgactt cgggctacgg ctgagagagt taaggccctg gagggtgcac tgaaggaggc
     3121 caaggagggc gctatgaagg acaagcgtag ataccagcag gaggtggacc gcatcaaaga
     3181 agccgtgcgg tacaagagct ccggcaagcg gggccattct gcccagatcg ctaagcctgt
     3241 gaggcctggc cactatcctg cctcctcacc caccaacccc tacggcaccc ggagccccga
     3301 gtgtatcagc tacaccaaca acctcttcca gaactaccag aacctgcacc tgcaggctgc
     3361 gcctagctcc acttcagata tgtactttgc cagcagcgga gccacatctg ttgccccctt
     3421 ggcttcctac cagaaggcca acatggacaa tggaaatgcc acagatatca acgacaacag
     3481 gagtgacctg ccgtgtggct atgaggctga ggaccaggcc aagcttttcc ctctccacca
     3541 agagacagca gccagcgaat tcgataagga ttgtgaaatg aagcgcacaa ccctggactc
     3601 cccactgggc aaacttgagc tgtctggatg cgagcaggga ctgcacagaa tcatattcct
     3661 cggaaagggc accagcgccg ctgatgcagt ggaagttcca gcaccagctg cagtgcttgg
     3721 tggaccagag ccacttatgc aggctacagc ttggctgaat gcttatttcc accagccgga
     3781 agccatcgaa gagtttcccg tgccagcact gcaccatcct gtctttcaac aagagtcctt
     3841 cacacggcaa gtcctgtgga aactcctgaa agtcgtgaaa tttggggaag tgatctccta
     3901 cagccacctg gctgcactgg ccggaaatcc tgcagcaact gccgctgtga aaactgccct
     3961 gtccggcaat ccagtgccaa tcctgattcc ttgtcaccgg gtcgtccagg gcgacctgga
     4021 tgttggagga tatgaaggcg gactggctgt caaagagtgg ctccttgctc acgagggcca
     4081 tagactcgga aagcctggac ttggtggaag cgccggatct gcagcaggat ctggcgagtt
     4141 tcaccaccac catcaccacg actacaagga cgacgacgac aaatgagcgg ccgctcgagt
     4201 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     4261 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     4321 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     4381 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     4441 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     4501 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4561 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4621 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4681 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4741 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4801 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4861 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4921 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     4981 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     5041 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     5101 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     5161 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     5221 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     5281 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     5341 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     5401 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     5461 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     5521 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     5581 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggca aagtgccggg
     5641 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     5701 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     5761 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     5821 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     5881 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     5941 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     6001 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     6061 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     6121 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     6181 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     6241 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     6301 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     6361 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6421 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     6481 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     6541 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     6601 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6661 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6721 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6781 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6841 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6901 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6961 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     7021 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     7081 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     7141 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     7201 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     7261 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     7321 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     7381 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7441 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7501 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     7561 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     7621 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7681 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7741 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7801 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7861 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     7921 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     7981 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     8041 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     8101 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     8161 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     8221 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     8281 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     8341 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     8401 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8461 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     8521 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     8581 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     8641 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     8701 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     8761 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
     8821 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
     8881 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     8941 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     9001 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     9061 attgacgtca atgggtggag
//
