LOCUS       sequence-396768-        5639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses H5_fold-0_Chantal in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5639)
  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 5639)
  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 _Chantal
FEATURES             Location/Qualifiers
     source          1..5639
                     /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             447..469
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    484..508
                     /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(508..527)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(509..527)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(611..630)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        836..913
                     /gene="lacI"
                     /label=lacI promoter
     CDS             914..1996
                     /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(933..952)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2805..2996
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2990..3012)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3098..3237
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3153..3172
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3252..3269)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3423..4011)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3503..3522)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4182..4973,4974..5042))
                     /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     4805..4824
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5043..5147)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5173..5628)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5310..5331)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5522..5541
                     /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 gaggctacct tcctctttct
      181 cgatcttttc gatcactttc aaggcttctg gggtaatgcc agcgcgtttc aggagttcct
      241 ctttcccatg tttctccgca taccggcgta acgcttcgac aacacgcgaa cgactgcgac
      301 caacacgacg tgctgcttct tcaatggcat cgcgtaccca ttcttccggc gttccgttgc
      361 gtttcttttc gcgagcatct tcgtaggcct gcgcaaacag ttccaccagt ttgtcaatct
      421 ctttctcttc ttcgcccata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag
      481 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc
      541 gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
      601 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
      661 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
      721 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
      781 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
      841 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
      901 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
      961 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     1021 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     1081 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     1141 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     1201 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     1261 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     1321 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     1381 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     1441 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     1501 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     1561 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     1621 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     1681 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     1741 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     1801 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     1861 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     1921 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     1981 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
     2041 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     2101 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     2161 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga
     2221 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg
     2281 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac
     2341 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta
     2401 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa
     2461 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg
     2521 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct
     2581 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga
     2641 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta
     2701 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta
     2761 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa
     2821 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac
     2881 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg
     2941 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     3001 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     3061 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     3121 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     3181 gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     3241 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3301 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3361 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3421 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3481 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3541 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3601 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3661 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3721 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3781 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3841 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     3901 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3961 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4021 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4081 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4141 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4201 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4261 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4321 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4381 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4441 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     4501 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4561 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4621 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4681 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4741 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4801 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4861 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4921 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4981 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5041 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5101 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5161 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     5221 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     5281 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     5341 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     5401 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     5461 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     5521 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     5581 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
