LOCUS       sequence-435686-        6390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Strep-HaloTag fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6390)
  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 6390)
  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: Strep-Halo
FEATURES             Location/Qualifiers
     source          1..6390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      144..732
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     633..652
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     886..903
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1020..1041
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1056..1061,1062..1079,1080..1086)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1094..1110
                     /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)."
     primer_bind     1099..1121
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1118..1134
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1118..1134
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1183..1304
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1194..1213)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1248..1267
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1478..2272)
                     /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(1589..1608)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2199..2218
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2339..2668)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2353..2488
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2407..2426)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2653..2673
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2682..3110)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2792..2813)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3004..3023
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3156..3380
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3369..3386
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(4355..4378,4379..4414,4415..4438))
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     primer_bind     complement(4485..4504)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4486..4504)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4549..4752)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4578..4598)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4753..5132
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5304..5323
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5398..5502
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5503..5571,5572..6363)
                     /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     complement(5721..5740)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
       61 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
      121 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
      181 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      241 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
      301 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      361 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      421 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      481 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      541 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      601 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      661 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      721 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      781 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      841 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      901 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
      961 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     1021 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     1081 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     1141 tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt
     1201 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     1261 ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg ggcgaagaac
     1321 tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg
     1381 aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc
     1441 gtcgcttggt cggtcatttc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc
     1501 gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt
     1561 cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat
     1621 agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca
     1681 ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca
     1741 tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca
     1801 gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt
     1861 tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat
     1921 cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg
     1981 gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
     2041 cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat
     2101 tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc
     2161 ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc
     2221 tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg
     2281 atcctcatcc tgtctcttga tcagatccga aaatggatat acaagctccc gggagctttt
     2341 tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggcag
     2401 aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
     2461 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
     2521 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     2581 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     2641 gggactttcc acaccctaac tgacacacat tccacagaat taattcgcgt taaatttttg
     2701 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2761 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2821 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2881 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     2941 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3001 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3061 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc
     3121 cctagagccc cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc
     3181 atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
     3241 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
     3301 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
     3361 ctggcaacta gaaggcacag tcgaggctga tcagcgggtt taaacgggcc ctctagactc
     3421 gagcggccgc ttaatcgatg ccgcttccag cagcagatcc agccgtacgt atttcaagcg
     3481 tggaaagcca ccgcgcgatt tctgacccga tcagatccgg gttgtcttct tgcaagaggt
     3541 tgaggcccgg cccaatgtcc acggccttac agtttgggag gctcttagcg agtctagcgg
     3601 cttcggccgg aggaatcaag acccctggcg ttccccagaa cagaagtttg gggacagggg
     3661 actgatgaag ccagtccatg tactcttcca caagcgctac tatgttagct ggctcgccgg
     3721 caataggcaa ctcgttcggg aaccgccaca gaggctctcg gtctacgggg ttcaagaaag
     3781 gttctcgata gtggtccatt tcgacttctg taagtggccg gacgactccc ataggaagcg
     3841 taccttcgat aaaaacattc tgatcgatta tcaatttgcg cccgacatca gttgtgcgga
     3901 atgcctggaa agtttctcgc gcgaattcgg gccactcgtc ccacgtgggt attggtcgta
     3961 taaattccat aaacgctatc cccttaactc tttctggatt tcttttcgcc caatggaatc
     4021 caagggcgga accccagtcg tggatcacca gcaccacctc ctcgagtccc agagcctcaa
     4081 tgaacgcgtc catgaatcga acgtggtcgt cgaaaaaata acccagatcg ggtttgtcgg
     4141 acttacccat gccgatcaaa tcgggtgcta tacacctatg ggtaggggcc acgtgtggga
     4201 ttatgtttct ccagacgtaa cttgatgtcg ggttgccatg cagaaacaat actggtgtac
     4261 cgtccctcgg tccgacatcc acataatgca ttcgctcacc caggacttca acgtaatgtg
     4321 ggtcaaaagg gaatcctgtg cctatggcgc ttcccttttc gaattgtggg tgactccagg
     4381 cacttccgcc agagccgcct ccagaaccgc cgcctttctc aaattgggga tggctccatc
     4441 ccatggtacc aagcttaagt ttaaacgcta gccagcttgg gtctccctat agtgagtcgt
     4501 attaatttcg ataagccagt aagcagtggg ttctctagtt agccagagag ctctgcttat
     4561 atagacctcc caccgtacac gcctaccgcc catttgcgtc aatggggcgg agttgttacg
     4621 acattttgga aagtcccgtt gattttggtg ccaaaacaaa ctcccattga cgtcaatggg
     4681 gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga tgtactgcca
     4741 aaaccgcatc accatggtaa tagcgatgac taatacgtag atgtactgcc aagtaggaaa
     4801 gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt cattgacgtc
     4861 aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg cagtttaccg
     4921 taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta tgggaacata
     4981 cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac
     5041 cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc ccgtaattga
     5101 ttactattaa taactagtca ataatcaatg tcaacgcgta tatctggccc gtacatcgcg
     5161 aagcagcgca aaacgcctaa ccctaagcag attcttcatg caattgtcgg tcaagccttg
     5221 ccttgttgta gcttaaattt tgctcgcgca ctactcagcg acctccaaca cacaagcagg
     5281 gagcagatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatag
     5341 gggatcggga gatctcccga tccgtcgacg tcaggtggca cttttcgggg aaatgtgcgc
     5401 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5461 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5521 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5581 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5641 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5701 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5761 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5821 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5881 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5941 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6001 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6061 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6121 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6181 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6241 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6301 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6361 taactgtcag accaagttta ctcatatata
//
