LOCUS       sequence-372292-        7648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of His6-TEV-SNAP-PRC1 in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7648)
  AUTHORS   Subramanian R, Ti SC, Tan L, Darst SA, Kapoor TM
  TITLE     Marking and measuring single microtubules by PRC1 and kinesin-4.
  JOURNAL   Cell. 2013 Jul 18;154(2):377-90. doi: 10.1016/j.cell.2013.06.021.
  PUBMED    23870126
REFERENCE   2  (bases 1 to 7648)
  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"; doi: 
            "10.1016/j.cell.2013.06"; journalName: "Cell"; date: "2013-07-18- 
            18"; volume: "154"; issue: "2"; pages: "377-90"
COMMENT     Sequence Label: His-TEV-SNAP-PRC1
FEATURES             Location/Qualifiers
     source          1..7648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             140..160
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             161..706
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             2597..2641
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2679..2697)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2693..2740
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2777..3232
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2864..2883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3074..3095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3347..4138,4139..4207))
                     /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     3970..3989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4208..4300)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4381..4969
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4870..4889
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5123..5140
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5155..5294
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5220..5239)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5380..5402
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5396..5587)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6159..7241)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7203..7222
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7242..7319)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7525..7544
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(7632..7648,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(7632..7648,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgga
      121 ttacgatatc ccaacgaccg aaaacctgta ttttcagggc atggacaaag attgcgaaat
      181 gaaacgtacc accctggata gcccgctggg caaactggaa ctgagcggct gcgaacaggg
      241 cctgcatgaa attaaactgc tgggtaaagg caccagcgcg gccgatgcgg ttgaagttcc
      301 ggccccggcc gccgtgctgg gtggtccgga accgctgatg caggcgaccg cgtggctgaa
      361 cgcgtatttt catcagccgg aagcgattga agaatttccg gttccggcgc tgcatcatcc
      421 ggtgtttcag caggagagct ttacccgtca ggtgctgtgg aaactgctga aagtggttaa
      481 atttggcgaa gtgattagct atcagcagct ggcggccctg gcgggtaatc cggcggccac
      541 cgccgccgtt aaaaccgcgc tgagcggtaa cccggtgccg attctgattc cgtgccatcg
      601 tgtggttagc tctagcggtg cggttggcgg ttatgaaggt ggtctggcgg tgaaagagtg
      661 gctgctggcc catgaaggtc atcgtctggg taaaccgggt ctgggaaagc ttgcggccgc
      721 aatgaggaga agtgaggtgc tggcggagga gtccatagta tgtctgcaga aagccctaaa
      781 tcaccttcgg gaaatatggg agctaattgg gattccagag gaccagcggt tacaaagaac
      841 tgaggtggta aagaagcata tcaaggaact cctggatatg atgattgctg aagaggaaag
      901 cctgaaggaa agactcatca aaagcatatc cgtctgtcag aaagagctga acactctgtg
      961 cagcgagtta catgttgagc catttcagga agaaggagag acaaccatct tgcaactaga
     1021 aaaagatttg cgcacccaag tggaattgat gcgaaaacag aaaaaggaga gaaaacagga
     1081 actgaagcta cttcaagagc aagatcaaga actgtgcgaa attctttgta tgccccacta
     1141 tgatattgac agtgcctcag tgcccagctt agaagagctg aaccagttca ggcaacatgt
     1201 gacaactttg agggaaacaa aggcttctag gcgtgaggag tttgtcagta taaagagaca
     1261 gatcatactg tgtatggaag aattagacca caccccagac acaagctttg aaagagatgt
     1321 ggtgtgtgaa gacgaagatg ccttttgttt gtctttggag aatattgcaa cactacaaaa
     1381 gttgctacgg cagctggaaa tgcagaaatc acaaaatgaa gcagtgtgtg aggggctgcg
     1441 tactcaaatc cgagagctct gggacaggtt gcaaatacct gaagaagaaa gagaagctgt
     1501 ggccaccatt atgtctgggt caaaggccaa ggtccggaaa gcgctgcaat tagaagtgga
     1561 tcggttggaa gaactgaaaa tgcaaaacat gaagaaagtg attgaggcaa ttcgagtgga
     1621 gctggttcag tactgggacc agtgctttta tagccaggag cagagacaag cttttgcccc
     1681 tttctgtgct gaggactaca cagaaagtct gctccagctc cacgatgctg agattgtgcg
     1741 gttaaaaaac tactatgaag ttcacaagga actctttgaa ggtgtccaga agtgggaaga
     1801 aacctggagg cttttcttag agtttgagag aaaagcttca gatccaaatc gatttacaaa
     1861 ccgaggagga aatcttctaa aagaagaaaa acaacgagcc aagctccaga aaatgctgcc
     1921 caagctggaa gaagagttga aggcacgaat tgaattgtgg gaacaggaac attcaaaggc
     1981 atttatggtg aatgggcaga aattcatgga gtatgtggca gaacaatggg agatgcatcg
     2041 attggagaaa gagagagcca agcaggaaag acaactgaag aacaaaaaac agacagagac
     2101 agagatgctg tatggcagcg ctcctcgaac acctagcaag cggcgaggac tggctcccaa
     2161 tacaccgggc aaagcacgta agctgaacac taccaccatg tccaatgcta cggccaatag
     2221 tagcattcgg cctatctttg gagggacagt ctaccactcc cccgtgtctc gacttcctcc
     2281 ttctggcagc aagccagtcg ctgcttccac ctgttcaggg aagaaaacac cccgtactgg
     2341 caggcatgga gccaacaagg agaacctgga gctcaacggc agcatcctga gtggtgggta
     2401 ccctggctcg gcccccctcc agcgcaactt cagcattaat tctgttgcca gcacctattc
     2461 tgagtttgcg aaggatccgt ccctctctga cagttccact gttgggcttc agcgagaact
     2521 ttcaaaggct tccaaatctg atgctacttc tggaatcctc aattcaacca acatccagtc
     2581 ctgactcgag tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc
     2641 gtctactagc gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata
     2701 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     2761 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2821 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2881 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2941 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3001 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3061 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3121 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3181 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat ttctggcggc
     3241 acgatggcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3301 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3361 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3421 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3481 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3541 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3601 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3661 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3721 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3781 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3841 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3901 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3961 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4021 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4081 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4141 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4201 tactcatact cttccttttt caatcatgat tgaagcattt atcagggtta ttgtctcatg
     4261 agcggataca tatttgaatg tatttagaaa aataaacaaa taggtcatga ccaaaatccc
     4321 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4381 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4441 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4501 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4561 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4621 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4681 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4741 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4801 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4861 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4921 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4981 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5041 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     5101 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     5161 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     5221 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     5281 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5341 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5401 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     5461 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     5521 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     5581 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     5641 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     5701 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     5761 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     5821 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     5881 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     5941 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     6001 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg
     6061 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     6121 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6181 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     6241 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     6301 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     6361 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     6421 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     6481 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     6541 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     6601 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     6661 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     6721 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     6781 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     6841 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     6901 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     6961 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     7021 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     7081 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     7141 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     7201 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     7261 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     7321 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     7381 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     7441 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     7501 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     7561 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga
     7621 tcccgcgaaa ttaatacgac tcactata
//
