LOCUS       sequence-370403-        7066 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-Sumo-CylK-Strep in E. coli for refolding protein 
            preparation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7066)
  AUTHORS   Braffman NR, Ruskoski TB, Davis KM, Glasser NR, Johnson C, Okafor 
            CD, Boal AK, Balskus EP
  TITLE     Structural basis for an unprecedented enzymatic alkylation in 
            cylindrocyclophane biosynthesis.
  JOURNAL   Elife. 2022 Feb 25;11. pii: 75761. doi: 10.7554/eLife.75761.
  PUBMED    35212625
REFERENCE   2  (bases 1 to 7066)
  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: "Elife."; 
            date: "2022-02-25"; volume: "11. pii: 75761. doi"; pages: " 
            10.7554/eLife.75761"
COMMENT     Sequence Label: pET-His6-Sumo-CylK-Strep
FEATURES             Location/Qualifiers
     source          1..7066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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     complement(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2014..2153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2079..2098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2239..2261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2255..2446)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3904..3923
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4007..4026
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4007..4025
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4114..4136
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4156..4173
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4192..4485
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             6520..6543
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(6602..6620)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6616..6663
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7029..7034,7035..7051,7052..7057))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2041 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     2101 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     2161 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2221 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2281 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     2341 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     2401 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     2461 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     2521 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     2581 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     2641 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     2701 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     2761 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     2821 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     2881 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     2941 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     3001 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     3061 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     3121 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     3181 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     3241 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     3301 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     3361 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     3421 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3481 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3541 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     3601 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     3661 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgac gctctccctt
     3721 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     3781 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     3841 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     3901 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     3961 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     4021 atagggagac cacaacggtt tccctctagt gccggctccg gagagctctt taattaagcg
     4081 gccgccctgc aggactcgag ttctagaaat aattttgttt aactttaaga aggagatata
     4141 catatgaaat cttctcacca tcaccatcac catggttctt ctatggctag catgtcggac
     4201 tcagaagtca atcaagaagc taagccagag gtcaagccag aagtcaagcc tgagactcac
     4261 atcaatttaa aggtgtccga tggatcttca gagatcttct tcaagatcaa aaagaccact
     4321 cctttaagaa ggctgatgga agcgttcgct aaaagacagg gtaaggaaat ggactcctta
     4381 agattcttgt acgacggtat tagaattcaa gctgatcaga cccctgaaga tttggacatg
     4441 gaggataacg atattattga ggctcacaga gaacagattg gtgggatgaa aaaaaacaaa
     4501 aaaacaacaa aatccttact ctcagccgac gaaaaaataa cagagtcctt acgctcaaca
     4561 ttgagtgacg ttcttccaga ccagcttcaa acatatatca gaactgtctt acaattctct
     4621 ggtagaccgg agggggccaa tctcctaaca ggccccaata ctgagataga atttttcagt
     4681 caagatccca ataagaattt tcccaacatt tttgccaagt atagtaatgt actgacagtt
     4741 tccagcgatc ctaatttcat aacatcagaa gatgaagagg tcaaaatcat ttggggccgt
     4801 catggaagtg atagcctaat tggttttgac cctggtgctg accttgttgg taaacgcaga
     4861 attgatattt tcctcggtga ttttatcgat gaacaattca atccaatacc tggcgcattg
     4921 aacgcaggta aatcgtggtc agacagattt attctgggtg attggcaaaa accctactat
     4981 tttgaggatg acgaaacctt gggtttaaac caatcagcta tgattttaga ctttaacccc
     5041 aatgaagatg tcattcagct ccacggcgat cgccaagact atgaactggt aaatattagc
     5101 ttgggaacag caatattttg gcgggagaaa aaaggttatg acctcatcgg cgttttaggt
     5161 ggagtctctg atttaagttt aaagggtgac tacttcgagt tcaagggcaa caccgcacca
     5221 aaaacagtcc taaagacggc tgaacatatt gggactgccg cgaatgatta catatttagt
     5281 tccaccgtag atgctaaggg caatttctat gttggaggag gaaccggggg atctttagga
     5341 ggacgcaaca taggagctag ggacgcttgg ctggccaaat acgatagcaa tggcaaccag
     5401 cgatggagta gacaatttgg ctcaaccggc actgaaagct tatggggtat ggctagtgac
     5461 gggagcaata tttacgttgc gggaaacacc acaggccagc tagaaaataa caccgttaaa
     5521 ggcggcaacg atgcttattt agctaaatac gatagtgatg gcaaccaagt gtggattaaa
     5581 cagaatggga cttacacact tgaggaatct tataaaatta ccgtcgattc cagtggcaat
     5641 atttacactg ctggtcacac ttttggcagc ttgggtgggc caaaccaaaa cttggaacaa
     5701 ggcgaagtat tcgagttacc atcgactgat ggctacgtgg ccaagttcga cagcaatggt
     5761 aatcagttgt gggttgcaca gtttggtacg atcacacttg acgacaattg gggtgtcgca
     5821 gccgacaaca atggcaatgt cttcgctgga ggaaatacaa aaggcagttt cggagcaaag
     5881 aatactggga cagcgggaga atatgatgcc tggctagtta agttgaacaa agatggtcag
     5941 actgattggg ttagacagtt tggcactccg aattatgact ttatgtggga cattgaaacg
     6001 gatagtctag gcgatatcta tgcgaccgga tggactctgg gagacttggg gggaaaaaat
     6061 gccggctctt atgatgtctg gctggccaag tacaacacta atggcaacca gttgtggatc
     6121 aaacagtttg gcactagtga agatgatgca ccctttttag atgggataga tatagacgca
     6181 aatgacaata ttttcctgac gggaaataca aacggcaact taggcggagc taatgctgga
     6241 tcttatgatg cttgggcggc gaagtttgac aaggatggca atcagttatg gctcaaacag
     6301 tttggtaccc ccgactatga cactgctacc acggtaactg ccgttaattt cggtaaactt
     6361 tatgtttcgg gaattactga gggaagtttg ggaactacca atgctgggtc ttatgactct
     6421 tgggcgctca aactagacgc tgataacgga gagatacaag attttaattc ttctactaat
     6481 acttttggcc aaacgggttt tttgaattta gggagcgctt ggagccaccc gcagttcgaa
     6541 aaataacgcg ctgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     6601 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     6661 ttgctgaaag gaggaactat atccggatat ccacaggacg ggtgtggtcg ccatgatcgc
     6721 gtagtcgata gtggctccaa gtagcgaagc gagcaggact gggcggcggc caaagcggtc
     6781 ggacagtgct ccgagaacgg gtgcgcatag aaattgcatc aacgcatata gcgctagcag
     6841 cacgccatag tgactggcga tgctgtcgga atggacgata tcccgcaaga ggcccggcag
     6901 taccggcata accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat
     6961 gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa
     7021 actaccgcat taaagcttat cgatgataag ctgtcaaaca tgagaa
//
