LOCUS       sequence-396706-        5944 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses H5_fold-0_Elsa in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5944)
  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 5944)
  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: H5_fold-0_Elsa
FEATURES             Location/Qualifiers
     source          1..5944
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(186..233)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     229..247
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(588..608)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(609..626)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             639..661
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    676..700
                     /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(700..719)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(701..719)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(803..822)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1028..1105
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1125..1144)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3499..3690
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3684..3706)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3792..3931
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3847..3866
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3946..3963)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4117..4705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4197..4216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4876..5667,5668..5736))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDDKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5499..5518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5737..5841)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5909..5927
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttagctttaa tgcggtagtt tatcacagtt aaattgctaa cgcagtcagg caccgtgtat
       61 gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc accctggatg ctgtaggcat
      121 aggcttggtt atgccggtac tgccgggcct cttgcgggat atccggatat agttcctcct
      181 ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc tagttattgc
      241 tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc cggatcctac
      301 tcgaggctgc cttcttcttt gcgaatctgc tgaatgactt tgtagatctc tggcgtaatg
      361 cctaagcgtt tcagcagttc atcgcgacct tgttcctgcg catatttggt gagcgcttca
      421 accacgtctt tccgcgaacg tccttgttgg cgtgcagcct cttctacggc atcacgtgcc
      481 cattcttccg gagtaccgtt cttcttctgg cgtttcgctt cacggaaagc ttcggcaaac
      541 agctccacca aggtttcgat ctctttctgc tcttccccca tatgtccgcc ctgaaaatac
      601 aggttttcgt gatggtgatg gtgatggccc atggtatatc tccttcttaa agttaaacaa
      661 aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa
      721 tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc
      781 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
      841 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
      901 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
      961 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     1021 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     1081 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     1141 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     1201 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     1261 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     1321 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     1381 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     1441 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     1501 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     1561 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     1621 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     1681 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     1741 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     1801 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     1861 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     1921 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     1981 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     2041 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     2101 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2161 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     2221 ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     2281 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     2341 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
     2401 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
     2461 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
     2521 ggcggccgac gcgctgggct acgtcttgct ggcgttcgcg acgcgaggct ggatggcctt
     2581 ccccattatg attcttctcg cttccggcgg catcgggatg cccgcgttgc aggccatgct
     2641 gtccaggcag gtagatgacg accatcaggg acagcttcaa ggatcgctcg cggctcttac
     2701 cagcctaact tcgatcattg gaccgctgat cgtcacggcg atttatgccg cctcggcgag
     2761 cacatggaac gggttggcat ggattgtagg cgccgcccta taccttgtct gcctccccgc
     2821 gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg gcggcacctc
     2881 gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag aactgtgaat
     2941 gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag cagccgcacg
     3001 cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg
     3061 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     3121 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     3181 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     3241 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     3301 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     3361 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     3421 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     3481 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     3541 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     3601 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg
     3661 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     3721 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     3781 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     3841 actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca
     3901 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     3961 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4021 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4081 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4141 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4201 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4261 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4321 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4381 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4441 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4501 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     4561 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4621 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4681 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4741 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4801 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4861 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4921 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4981 cttatcatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5041 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5101 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5161 taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt
     5221 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5281 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5341 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5401 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5461 gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag
     5521 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5581 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5641 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5701 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5761 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5821 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     5881 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca
     5941 agaa
//
