LOCUS       sequence-396772-        5753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses H7_fold-K _Mussoc in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5753)
  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 5753)
  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: H7_fold-K _Mussoc
FEATURES             Location/Qualifiers
     source          1..5753
                     /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             561..583
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    598..622
                     /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(622..641)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(623..641)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(725..744)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        950..1027
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1028..2110
                     /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(1047..1066)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2919..3110
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3104..3126)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3212..3351
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3267..3286
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3366..3383)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3537..4125)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3617..3636)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4296..5087,5088..5156))
                     /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     4919..4938
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5157..5261)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5287..5742)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5424..5445)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5636..5655
                     /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 gaggctccct ttctccttct
      181 tttcctggcg tgctttcttg atcgcttcct gtaaggcggt ttgccactga ggcgagtttg
      241 ccagtttctg gagctgcgga gatggcgctt tcgacagcgc tttgcgaatc cgttcaatcg
      301 cttcttcgat ttcctccgct gatgcaccgc tttctttcag ggcacgaagc agagtttcgg
      361 caaaacggcg agcagctttc tccagacgtt cgctatcgcc ttgctgttgc gctaacgcgg
      421 cctcagcgat ttcttccgcc aagcgtttca gggtttccgg cgtaattttg ccctgtttct
      481 cgaattcgga cagggcttca ctcaaggcac gttctgcttc gcgcttcagt ttctctttgt
      541 cctcatcacc catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga
      601 attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc
      661 tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct
      721 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg
      781 agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc
      841 atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg
      901 ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga
      961 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     1021 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     1081 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     1141 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     1201 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     1261 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     1321 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     1381 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     1441 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     1501 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     1561 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     1621 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     1681 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     1741 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     1801 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
     1861 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     1921 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     1981 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     2041 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     2101 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc
     2161 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
     2221 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg
     2281 cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc
     2341 tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg
     2401 ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc
     2461 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
     2521 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
     2581 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     2641 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     2701 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     2761 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     2821 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     2881 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     2941 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     3001 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     3061 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     3121 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     3181 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     3241 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     3301 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     3361 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3421 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3481 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3541 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3601 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3661 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3721 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3781 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3841 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3901 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3961 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4021 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4081 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4141 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4201 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4261 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4321 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4381 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4441 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4501 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4561 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
     4621 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4681 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4741 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4801 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4861 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4921 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4981 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5041 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5101 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5161 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5221 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5281 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     5341 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     5401 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     5461 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     5521 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     5581 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     5641 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     5701 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca
//
