LOCUS       sequence-332010-        7822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of His6-TEV-EGFP-PRC1 in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7822)
  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 7822)
  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-EGFP-PRC1
FEATURES             Location/Qualifiers
     source          1..7822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..88
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(82..273)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(845..1927)
                     /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     1889..1908
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1928..2005)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2211..2230
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2318..2337
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2318..2336
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2337..2361
                     /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             2376..2398
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2417..2434
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2474..2494
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2504..2506,2507..2509,2510..3220)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2504..2506,2507..2509,2510..3220)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2549..2570)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2810..2829)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3157..3178
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5105..5149
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5187..5205)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5201..5248
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5285..5740
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5372..5391)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5582..5603
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5855..6646,6647..6715))
                     /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     6478..6497
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6716..6808)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6889..7477
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7378..7397
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7631..7648
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7663..7802
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7728..7747)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
       61 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
      121 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
      181 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
      241 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
      301 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
      361 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
      421 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
      481 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
      541 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
      601 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
      661 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
      721 cacgatcatg ctagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
      781 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
      841 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
      901 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
      961 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     1021 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     1081 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     1141 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     1201 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     1261 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     1321 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     1381 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     1441 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     1501 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     1561 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     1621 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     1681 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     1741 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     1801 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     1861 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     1921 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     1981 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     2041 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     2101 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     2161 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     2221 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     2281 gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     2341 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
     2401 taccatgggc agcagccatc accatcatca ccacagccag gatccgaatt cggattacga
     2461 tatcccaacg accgaaaacc tgtattttca gggcgcggcc gcaatggtga gcaagggcga
     2521 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     2581 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     2641 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     2701 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     2761 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     2821 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     2881 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     2941 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     3001 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     3061 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     3121 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     3181 cgccgccggg atcactctcg gcatggacga gctgtacaag gcggccgcaa tgaggagaag
     3241 tgaggtgctg gcggaggagt ccatagtatg tctgcagaaa gccctaaatc accttcggga
     3301 aatatgggag ctaattggga ttccagagga ccagcggtta caaagaactg aggtggtaaa
     3361 gaagcatatc aaggaactcc tggatatgat gattgctgaa gaggaaagcc tgaaggaaag
     3421 actcatcaaa agcatatccg tctgtcagaa agagctgaac actctgtgca gcgagttaca
     3481 tgttgagcca tttcaggaag aaggagagac aaccatcttg caactagaaa aagatttgcg
     3541 cacccaagtg gaattgatgc gaaaacagaa aaaggagaga aaacaggaac tgaagctact
     3601 tcaagagcaa gatcaagaac tgtgcgaaat tctttgtatg ccccactatg atattgacag
     3661 tgcctcagtg cccagcttag aagagctgaa ccagttcagg caacatgtga caactttgag
     3721 ggaaacaaag gcttctaggc gtgaggagtt tgtcagtata aagagacaga tcatactgtg
     3781 tatggaagaa ttagaccaca ccccagacac aagctttgaa agagatgtgg tgtgtgaaga
     3841 cgaagatgcc ttttgtttgt ctttggagaa tattgcaaca ctacaaaagt tgctacggca
     3901 gctggaaatg cagaaatcac aaaatgaagc agtgtgtgag gggctgcgta ctcaaatccg
     3961 agagctctgg gacaggttgc aaatacctga agaagaaaga gaagctgtgg ccaccattat
     4021 gtctgggtca aaggccaagg tccggaaagc gctgcaatta gaagtggatc ggttggaaga
     4081 actgaaaatg caaaacatga agaaagtgat tgaggcaatt cgagtggagc tggttcagta
     4141 ctgggaccag tgcttttata gccaggagca gagacaagct tttgcccctt tctgtgctga
     4201 ggactacaca gaaagtctgc tccagctcca cgatgctgag attgtgcggt taaaaaacta
     4261 ctatgaagtt cacaaggaac tctttgaagg tgtccagaag tgggaagaaa cctggaggct
     4321 tttcttagag tttgagagaa aagcttcaga tccaaatcga tttacaaacc gaggaggaaa
     4381 tcttctaaaa gaagaaaaac aacgagccaa gctccagaaa atgctgccca agctggaaga
     4441 agagttgaag gcacgaattg aattgtggga acaggaacat tcaaaggcat ttatggtgaa
     4501 tgggcagaaa ttcatggagt atgtggcaga acaatgggag atgcatcgat tggagaaaga
     4561 gagagccaag caggaaagac aactgaagaa caaaaaacag acagagacag agatgctgta
     4621 tggcagcgct cctcgaacac ctagcaagcg gcgaggactg gctcccaata caccgggcaa
     4681 agcacgtaag ctgaacacta ccaccatgtc caatgctacg gccaatagta gcattcggcc
     4741 tatctttgga gggacagtct accactcccc cgtgtctcga cttcctcctt ctggcagcaa
     4801 gccagtcgct gcttccacct gttcagggaa gaaaacaccc cgtactggca ggcatggagc
     4861 caacaaggag aacctggagc tcaacggcag catcctgagt ggtgggtacc ctggctcggc
     4921 ccccctccag cgcaacttca gcattaattc tgttgccagc acctattctg agtttgcgaa
     4981 ggatccgtcc ctctctgaca gttccactgt tgggcttcag cgagaacttt caaaggcttc
     5041 caaatctgat gctacttctg gaatcctcaa ttcaaccaac atccagtcct gactcgagtc
     5101 tggtaaagaa accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc
     5161 agcttaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     5221 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     5281 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5341 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     5401 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5461 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     5521 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5581 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5641 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5701 tttaacgcga attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga
     5761 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5821 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5881 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5941 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6001 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6061 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6121 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6181 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6241 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6301 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6361 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6421 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6481 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6541 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6601 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6661 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6721 tcctttttca atcatgattg aagcatttat cagggttatt gtctcatgag cggatacata
     6781 tttgaatgta tttagaaaaa taaacaaata ggtcatgacc aaaatccctt aacgtgagtt
     6841 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6901 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6961 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7021 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7081 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7141 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7201 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7261 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7321 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7381 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7441 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7501 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     7561 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     7621 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     7681 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     7741 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     7801 cgccccgaca cccgccaaca cc
//
