LOCUS       sequence-430247-        6398 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression of RAN with an N-terminal 6His tag and a TEV 
            cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6398)
  AUTHORS   Pardal AJ, Bowman AJ
  TITLE     A specific role for importin-5 and NASP in the import and nuclear 
            hand-off of monomeric H3.
  JOURNAL   Elife. 2022 Sep 6;11:e81755. doi: 10.7554/eLife.81755.
  PUBMED    36066346
REFERENCE   2  (bases 1 to 6398)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2022-09-6"; volume: "11:e81755. doi"; pages: " 
            10.7554/eLife.81755"
COMMENT     Sequence Label: pCL1333
FEATURES             Location/Qualifiers
     source          1..6398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(674..694)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(716..733)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             752..774
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    789..813
                     /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     complement(813..832)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(814..832)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(916..935)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1141..1218
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1238..1257)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3612..3803
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3797..3819)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3905..4044
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3960..3979
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4059..4076)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4230..4818)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4310..4329)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4989..5780,5781..5849))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5612..5631
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5850..5954)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6022..6040
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(6067..6072,6073..6089,6090..6095)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(6270..6317)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6313..6331
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 actattagga tcctcacagg tcatcatcct catccgggag agcagttgtc tgagcaacct
       61 ctaagtcgtg ctcatactgt gctgccaaag ctgggtccat gacaacttct ggtggggcga
      121 gagcaggcat ggcaacaaat tccaagttag ggtctccaat gagcttccta gcaagccaga
      181 ggaagggctt ttcaaagttg tagttacttt tggcagaaat gtcgtagtac tgaagattct
      241 tctttcggtg gaagacaatg gatttcgcct tcactttcct gtccttaata tccactttgt
      301 tgccacacaa cacaatgggg atgttttcac acactcgtac cagatctcta tgccagttag
      361 gcacattctt gtaagtaact ctcgatgtta catcaaacat tatgatggca cactgggctt
      421 ggatataata gccatctctc agtccaccga atttctcctg gccggctgtg tcccatacat
      481 tgaacttaat aggtcctctg ttggtgtgga acactagggg atgaacctca acacccaagg
      541 tggctacata cttcttctca aattcaccag tcaaatgacg tttcacgaag gtcgtttttc
      601 cagtaccacc atcaccaacc aatacaagtt tgaactggac ctggggctct ccctgcgcag
      661 ccatcgccat atggccctgg aagtaaaggt tttcgccgct gccggtaccg ctgctgtgat
      721 gatgatgatg atggctgctg cccatggtat atctccttct taaagttaaa caaaattatt
      781 actagtgggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
      841 ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
      901 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
      961 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     1021 tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca
     1081 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg
     1141 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     1201 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     1261 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     1321 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     1381 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     1441 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     1501 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     1561 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     1621 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     1681 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     1741 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     1801 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     1861 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     1921 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     1981 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     2041 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     2101 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     2161 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     2221 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acagtttccc
     2281 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
     2341 ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     2401 cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga
     2461 caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg
     2521 atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc
     2581 actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc
     2641 gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag gctggatggc cttccccatt
     2701 atgattcttc tcgcttccgg cggcatcggg atgcccgcgt tgcaggccat gctgtccagg
     2761 caggtagatg acgaccatca gggacagctt caaggatcgc tcgcggctct taccagccta
     2821 acttcgatca ttggaccgct gatcgtcacg gcgatttatg ccgcctcggc gagcacatgg
     2881 aacgggttgg catggattgt aggcgccgcc ctataccttg tctgcctccc cgcgttgcgt
     2941 cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcggcac ctcgctaacg
     3001 gattcaccac tccaagaatt ggagccaatc aattcttgcg gagaactgtg aatgcgcaaa
     3061 ccaacccttg gcagaacata tccatcgcgt ccgccatctc cagcagccgc acgcggcgca
     3121 tctcgggcag cgttgggtcc tggccacggg tgcgcatgat cgtgctcctg tcgttgagga
     3181 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     3241 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     3301 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     3361 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     3421 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     3481 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     3541 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     3601 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     3661 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     3721 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     3781 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3841 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3901 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     3961 ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc
     4021 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4081 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4141 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4201 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4261 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4321 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4381 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4441 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4501 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4561 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4621 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4681 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4741 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4801 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4861 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4921 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4981 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5041 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5101 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5161 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5221 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5281 ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg
     5341 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5401 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5461 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5521 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5581 ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta
     5641 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5701 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5761 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5821 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5881 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5941 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     6001 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc
     6061 tcatgtttga cagcttatca tcgataagct ttaatgcggt agtttatcac agttaaattg
     6121 ctaacgcagt caggcaccgt gtatgaaatc taacaatgcg ctcatcgtca tcctcggcac
     6181 cgtcaccctg gatgctgtag gcataggctt ggttatgccg gtactgccgg gcctcttgcg
     6241 ggatatccgg atatagttcc tcctttcagc aaaaaacccc tcaagacccg tttagaggcc
     6301 ccaaggggtt atgctagtta ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg
     6361 gctttgttag cagccggatc tcctagggct agctctag
//
