LOCUS       sequence-435644-        6039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of CLIPf-HisTag-FLAG fusion protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6039)
  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 6039)
  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: pCDNA3.1-CLIPf
FEATURES             Location/Qualifiers
     source          1..6039
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(90..109)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        281..660
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        661..864
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     815..835
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     909..928
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        909..927
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1587..1604
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1605..1628
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1676..1693)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1682..1906
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1952..2380
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2039..2058)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2249..2270
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2389..2409)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2394..2723
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2574..2709
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2636..2655
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2790..3584
                     /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(2844..2863)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3454..3473
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3758..3879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3795..3814)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3849..3868
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3928..3944)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3928..3944)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3941..3963)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3952..3968
                     /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(3976..3982,3983..4000,4001..4006))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4021..4042
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4159..4176)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4330..4918)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4410..4429)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5089..5880,5881..5949))
                     /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     5712..5731
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5950..6039,1..15))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
       61 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
      121 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
      181 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
      241 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
      301 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      361 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      421 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      481 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      541 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      601 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      661 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      721 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      781 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      841 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      901 atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt aaacttaagc
      961 ttggtaccat ggacaaggac tgcgagatga agcggaccac actggatagc cctctgggca
     1021 agctggaact gagcggctgt gaacagggcc tgcaccggat catctttctc ggcaagggaa
     1081 cctctgccgc cgatgctgtt gaagtgcctg ctcctgctgc tgtgctcgga ggacctgaac
     1141 ctctgattca ggccaccgcc tggctgaacg cctactttca tcagcccgag gccatcgagg
     1201 agttccccgt tcctgctctg catcaccccg tgttccagca agaaagcttc accagacagg
     1261 tgctgtggaa gctgctgaag gtggtcaagt tcggcgaagt gatcagcgag tctcacctgg
     1321 ccgctctcgt gggaaatcca gctgctacag ccgccgtgaa cacagccctg gatggaaacc
     1381 ctgtgcctat tctgatcccc tgccacagag tggtgcaggg cgattctgat gtgggccctt
     1441 atcttggagg cctggccgtg aaagaatggc tgctggccca cgaaggacac agactgggaa
     1501 aacctggcct cggaggatcc gctggatctg ctgctggaag cggcgaattc ggaagcgccg
     1561 gatctgcagc aggatctggc gagtttcacc accaccatca ccacgactac aaggacgacg
     1621 acgacaaatg agcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     1681 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1741 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1801 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1861 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     1921 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     1981 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2041 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2101 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2161 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2221 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2281 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2341 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     2401 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2461 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2521 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     2581 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     2641 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     2701 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     2761 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     2821 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     2881 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     2941 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3001 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3061 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3121 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3181 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3241 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3301 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3361 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3421 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3481 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3541 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     3601 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     3661 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     3721 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     3781 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3841 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     3901 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3961 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4021 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4081 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4141 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4201 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4261 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4321 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4381 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4441 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4501 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4561 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4621 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4681 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4741 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4801 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4861 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4921 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4981 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5041 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5101 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5161 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5221 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5281 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5341 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5401 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5461 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5521 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5581 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5641 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5701 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5761 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5821 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5881 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5941 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6001 gagcggatac atatttgaat gtatttagaa aaataaaca
//
