LOCUS       sequence-396769-        5705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses H6_fold-C _Rei in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5705)
  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 5705)
  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-C _Rei
FEATURES             Location/Qualifiers
     source          1..5705
                     /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             513..535
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    550..574
                     /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(574..593)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(575..593)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(677..696)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        902..979
                     /gene="lacI"
                     /label=lacI promoter
     CDS             980..2062
                     /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(999..1018)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2871..3062
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3056..3078)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3164..3303
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3219..3238
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3318..3335)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3489..4077)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3569..3588)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4248..5039,5040..5108))
                     /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     4871..4890
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5109..5213)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5239..5694)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5376..5397)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5588..5607
                     /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 gagggaacca cgttcagctt
      181 cttccatggc tttcttcagt tcctgtactt ccggattgtt cggttcctgt tcctccataa
      241 ccttcactgc ctgtttaaac gcaggcgatt tcagcacttt gcgcatgtct ttgcgagctt
      301 gctcttcgtt gccctgtttc cattcccgaa tggcttgctc aactttggtt ttcacctcgt
      361 ctttcgcttt ctggatctca tccggacttt tgccctgacg cttcagttct tctgccattg
      421 cttccagcag tttcttgaga agatctgggc ttttgcggac taactccaac gcacgttcgg
      481 cttgtttctc cgcttcatcc cccatatgta tatctccttc ttaaagttaa acaaaattat
      541 ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc
      601 gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
      661 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
      721 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
      781 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
      841 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
      901 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
      961 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     1021 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     1081 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     1141 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     1201 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     1261 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     1321 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     1381 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     1441 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     1501 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     1561 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     1621 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     1681 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     1741 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     1801 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     1861 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     1921 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     1981 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     2041 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     2101 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     2161 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     2221 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     2281 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     2341 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     2401 ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     2461 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     2521 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     2581 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     2641 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     2701 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     2761 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     2821 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     2881 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     2941 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     3001 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     3061 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3121 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3181 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     3241 gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3301 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3361 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3421 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3481 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3541 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3601 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3661 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3721 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3781 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3841 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3901 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     3961 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4021 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4081 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4141 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4201 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4261 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4321 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4381 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4441 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4501 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4561 gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4621 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4681 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4741 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4801 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4861 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4921 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4981 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5041 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5101 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5161 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5221 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     5281 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     5341 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     5401 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     5461 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     5521 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     5581 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     5641 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     5701 cgcca
//
