LOCUS       sequence-404966-        5708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses H6_fold-U _Nomur in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5708)
  AUTHORS   Sakuma K, Kobayashi N, Sugiki T, Nagashima T, Fujiwara T, Suzuki K, 
            Kobayashi N, Murata T, Kosugi T, Tatsumi-Koga R, Koga N
  TITLE     Design of complicated all-alpha protein structures.
  JOURNAL   Nat Struct Mol Biol. 2024 Feb;31(2):275-282. doi: 
            10.1038/s41594-023-01147-9. Epub 2024 Jan 4.
  PUBMED    38177681
REFERENCE   2  (bases 1 to 5708)
  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: 
            "10.1038/s41594-023-01147-9"; journalName: "Nat Struct Mol Biol"; 
            date: "2024-02"; volume: "31"; issue: "2"; pages: "275-282"
COMMENT     Sequence Label: H6_fold-U _Nomur
FEATURES             Location/Qualifiers
     source          1..5708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             516..538
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    553..577
                     /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(577..596)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(578..596)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(680..699)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        905..982
                     /gene="lacI"
                     /label=lacI promoter
     CDS             983..2065
                     /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     complement(1002..1021)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2874..3065
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3059..3081)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3167..3306
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3222..3241
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3321..3338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3492..4080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3572..3591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4251..5042,5043..5111))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4874..4893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5112..5216)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5242..5697)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5379..5400)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5591..5610
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gaggctcccg cgtttgcgaa
      181 cttcctcagc ttggcgagcc gcctctttag ccgcaccttc ttccgcaagc cgctctaatg
      241 cggacgccag tttcttgata atctccggac tcgcaccggc tgtacgtgca gcttcttcca
      301 ggagtttctt cgctttctcc gtgttgccat tcgacgcttc gcgaaaggct tcgaaaatct
      361 tttctgcggc ttgacgccac tgttccggag aggcggtttt cagcactttc tccagctctt
      421 ccagcgcttt ctgggcttct ggggtagtgg cctgttcgcg tgctgcacgc aacgcttcct
      481 gtgccgcttt ggctttggtt tcgcccatat gtatatctcc ttcttaaagt taaacaaaat
      541 tatttctaga ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt
      601 cgcgggatcg agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
      661 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
      721 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
      781 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
      841 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat
      901 cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
      961 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     1021 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     1081 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     1141 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     1201 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     1261 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     1321 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     1381 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     1441 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     1501 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     1561 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     1621 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     1681 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     1741 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     1801 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     1861 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     1921 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     1981 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     2041 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     2101 aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     2161 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     2221 taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg
     2281 cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct
     2341 tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg
     2401 cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg
     2461 gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct
     2521 gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt
     2581 aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag
     2641 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac
     2701 cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac
     2761 gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt
     2821 tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca
     2881 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc
     2941 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg
     3001 accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     3061 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     3121 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     3181 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     3241 actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     3301 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3361 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3421 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3481 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3541 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3601 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3661 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3721 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3781 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3841 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3901 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     3961 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4021 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4081 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4141 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4201 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4261 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4321 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4381 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4441 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4501 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4561 attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4621 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4681 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4741 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4801 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4861 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4921 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4981 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5041 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5101 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5161 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5221 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     5281 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     5341 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     5401 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     5461 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     5521 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     5581 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     5641 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     5701 attcgcca
//
