LOCUS       sequence-435692-        6582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning template for N-terminal CLIPf tagging and C-terminal SNAPf 
            tagging of constructs for mammalian expression. Also used for the 
            mammalian expression of CLIPf-SNAPf-HisTag-FLAG fusion protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6582)
  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 6582)
  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: pCLAP
FEATURES             Location/Qualifiers
     source          1..6582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(140..728)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(220..239)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(899..1690,1691..1759))
                     /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     1522..1541
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1760..1864)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1939..1958)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2130..2509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2510..2713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2664..2684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2758..2777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2758..2776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3979..3996
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3997..4020
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4068..4085)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4074..4298
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4344..4772
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4431..4450)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4641..4662
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4781..4801)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4786..5115
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4966..5101
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5028..5047
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5182..5976
                     /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(5236..5255)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5846..5865
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6150..6271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6187..6206)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6241..6260
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6320..6336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6320..6336)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6333..6355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6344..6360
                     /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(6368..6374,6375..6392,6393..6398))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6413..6434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6551..6568)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
       61 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
      121 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
      181 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      241 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      301 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      361 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      421 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      481 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      541 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
      601 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
      661 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
      721 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
      781 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
      841 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
      901 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
      961 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     1021 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     1081 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     1141 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     1201 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     1261 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     1321 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     1381 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     1441 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     1501 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     1561 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     1621 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     1681 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     1741 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     1801 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     1861 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     1921 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     1981 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     2041 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     2101 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     2161 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     2221 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     2281 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     2341 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     2401 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     2461 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     2521 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     2581 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     2641 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     2701 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     2761 tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct tggtaccatg
     2821 gacaaggact gcgagatgaa gcggaccaca ctggatagcc ctctgggcaa gctggaactg
     2881 agcggctgtg aacagggcct gcaccggatc atctttctcg gcaagggaac ctctgccgcc
     2941 gatgctgttg aagtgcctgc tcctgctgct gtgctcggag gacctgaacc tctgattcag
     3001 gccaccgcct ggctgaacgc ctactttcat cagcccgagg ccatcgagga gttccccgtt
     3061 cctgctctgc atcaccccgt gttccagcaa gaaagcttca ccagacaggt gctgtggaag
     3121 ctgctgaagg tggtcaagtt cggcgaagtg atcagcgagt ctcacctggc cgctctcgtg
     3181 ggaaatccag ctgctacagc cgccgtgaac acagccctgg atggaaaccc tgtgcctatt
     3241 ctgatcccct gccacagagt ggtgcagggc gattctgatg tgggccctta tcttggaggc
     3301 ctggccgtga aagaatggct gctggcccac gaaggacaca gactgggaaa acctggcctc
     3361 ggaggatccg ctggatctgc tgctggaagc ggcgaattcg ataaggattg tgaaatgaag
     3421 cgcacaaccc tggactcccc actgggcaaa cttgagctgt ctggatgcga gcagggactg
     3481 cacagaatca tattcctcgg aaagggcacc agcgccgctg atgcagtgga agttccagca
     3541 ccagctgcag tgcttggtgg accagagcca cttatgcagg ctacagcttg gctgaatgct
     3601 tatttccacc agccggaagc catcgaagag tttcccgtgc cagcactgca ccatcctgtc
     3661 tttcaacaag agtccttcac acggcaagtc ctgtggaaac tcctgaaagt cgtgaaattt
     3721 ggggaagtga tctcctacag ccacctggct gcactggccg gaaatcctgc agcaactgcc
     3781 gctgtgaaaa ctgccctgtc cggcaatcca gtgccaatcc tgattccttg tcaccgggtc
     3841 gtccagggcg acctggatgt tggaggatat gaaggcggac tggctgtcaa agagtggctc
     3901 cttgctcacg agggccatag actcggaaag cctggacttg gtggaagcgc cggatctgca
     3961 gcaggatctg gcgagtttca ccaccaccat caccacgact acaaggacga cgacgacaaa
     4021 tgagcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     4081 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4141 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4201 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4261 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     4321 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4381 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4441 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4501 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4561 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4621 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4681 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4741 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     4801 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4861 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4921 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4981 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     5041 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     5101 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     5161 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     5221 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     5281 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5341 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     5401 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5461 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5521 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5581 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5641 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5701 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5761 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5821 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5881 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5941 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     6001 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     6061 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     6121 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     6181 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     6241 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     6301 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     6361 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     6421 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6481 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     6541 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tc
//
