LOCUS       sequence-435683-        6036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of SNAPf-HisTag-FLAG fusion protein. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6036)
  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 6036)
  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-SNAPf
FEATURES             Location/Qualifiers
     source          1..6036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     581..600
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(819..923)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(998..1017)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1189..1568
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1569..1772
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1723..1743
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1817..1836
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1817..1835
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2492..2509
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2510..2533
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2581..2598)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2587..2811
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2857..3285
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2944..2963)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3154..3175
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3294..3314)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3299..3628
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3479..3614
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3541..3560
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3695..4489
                     /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(3749..3768)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4359..4378
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4663..4784
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4700..4719)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4754..4773
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4833..4849)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4833..4849)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4846..4868)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4857..4873
                     /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(4881..4887,4888..4905,4906..4911))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4926..4947
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5064..5081)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5235..5823)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5315..5334)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5994..6036,1..749,750..818))
                     /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"
ORIGIN
        1 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
       61 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
      121 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
      181 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
      241 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
      301 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
      361 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
      421 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
      481 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
      541 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
      601 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
      661 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
      721 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
      781 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
      841 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
      901 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
      961 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     1021 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     1081 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     1141 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     1201 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     1261 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     1321 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     1381 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     1441 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     1501 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     1561 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     1621 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     1681 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     1741 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt
     1801 actggcttat cgaaattaat acgactcact atagggagac ccaagctggc tagcgtttaa
     1861 acttaagctt ggtaccggat ccgctggatc tgctgctgga agcggcgaat tcgataagga
     1921 ttgtgaaatg aagcgcacaa ccctggactc cccactgggc aaacttgagc tgtctggatg
     1981 cgagcaggga ctgcacagaa tcatattcct cggaaagggc accagcgccg ctgatgcagt
     2041 ggaagttcca gcaccagctg cagtgcttgg tggaccagag ccacttatgc aggctacagc
     2101 ttggctgaat gcttatttcc accagccgga agccatcgaa gagtttcccg tgccagcact
     2161 gcaccatcct gtctttcaac aagagtcctt cacacggcaa gtcctgtgga aactcctgaa
     2221 agtcgtgaaa tttggggaag tgatctccta cagccacctg gctgcactgg ccggaaatcc
     2281 tgcagcaact gccgctgtga aaactgccct gtccggcaat ccagtgccaa tcctgattcc
     2341 ttgtcaccgg gtcgtccagg gcgacctgga tgttggagga tatgaaggcg gactggctgt
     2401 caaagagtgg ctccttgctc acgagggcca tagactcgga aagcctggac ttggtggaag
     2461 cgccggatct gcagcaggat ctggcgagtt tcaccaccac catcaccacg actacaagga
     2521 cgacgacgac aaatgagcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag
     2581 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2641 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2701 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2761 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     2821 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     2881 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2941 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3001 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3061 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3121 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3181 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3241 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     3301 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3361 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     3421 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3481 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     3541 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     3601 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     3661 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3721 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3781 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3841 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     3901 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3961 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4021 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4081 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4141 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4201 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4261 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4321 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4381 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4441 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4501 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4561 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4621 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     4681 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4741 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4801 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4861 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4921 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4981 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5041 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5101 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5161 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5221 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5281 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5341 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5401 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5461 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5521 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5581 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5641 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5701 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5761 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5821 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5881 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5941 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6001 tgcttaatca gtgaggcacc tatctcagcg atctgt
//
