LOCUS       sequence-221695-        6040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gHEEE_02 with a N-terminal 10-His DsbC TEV-peptide fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6040)
  AUTHORS   Buchko GW, Pulavarti SVSRK, Ovchinnikov V, Shaw EA, Rettie SA, Myler
            PJ, Karplus M, Szyperski T, Baker D, Bahl CD
  TITLE     Cytosolic expression, solution structures, and molecular dynamics 
            simulation of genetically encodable disulfide-rich de novo designed 
            peptides.
  JOURNAL   Protein Sci. 2018 Aug 26. doi: 10.1002/pro.3453.
  PUBMED    30152054
REFERENCE   2  (bases 1 to 6040)
  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"; journalName: "Protein 
            Sci. 2018 Aug 26. doi: 10.1002/pro.3453."
COMMENT     Sequence Label: pCDB367
FEATURES             Location/Qualifiers
     source          1..6040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..107
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     195..214
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        195..213
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    218..234
                     /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)."
     RBS             253..275
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             289..318
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             973..993
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(1165..1183)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1179..1226
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1263..1718
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1350..1369)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1560..1581
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1810..2625)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1810..2625)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2533..2552
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2617..2636
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2747..3335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3236..3255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3489..3506
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3521..3660
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3586..3605)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3746..3768
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3762..3953)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4762..5844)
                     /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     5806..5825
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5845..5922)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
       61 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
      121 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct
      181 cgatcccgcg aaattaatac gactcactat agggaaattg tgagcggata acaattcccc
      241 tctagaaata attttgttta actttaagaa ggagatatac atatgggtca ccatcatcac
      301 caccatcacc atcaccacgg tagcgcggca attcaacaaa cgttagccaa aatgggcatc
      361 aaaagcagcg atattcagcc cgcgcccgta gctggcatga agacagttct gactaacagc
      421 ggcgtgttgt acatcaccga tgatggtaaa catatcattc aggggccaat gtatgacgtt
      481 agcggcacgg ctccggtcaa tgtcaccaat aagatgctgt taaagcagtt gaatgcgctt
      541 gaaaaagaga tgatcgttta taaagcgccg caggaaaaac acgtcatcac cgtgtttact
      601 gatattacct gtggttactg ccacaaactg catgagcaaa tggcagacta taacgcgctg
      661 gggatcaccg tgcgttatct tgctttcccg cgccaggggc tggacagcga tgcagagaaa
      721 gaaatgaaag ctatctggtg tgcgaaagat aaaaacaaag cgtttgatga tgtgatggca
      781 ggtaaaagcg tcgcaccagc cagctgcgac gtggatattg ccgaccatta cgtacttggc
      841 gtccagcttg gcgttagcgg tactccggca gttgtgctga gcaatggcac acttgttccg
      901 ggttaccagc cgccgaaaga gatgaaagaa ttcctcgacg aacaccaaaa aatgaccagc
      961 ggtaaaggta gcgaaaacct gtattttcag tctcaggaaa cccgtaagaa atgcaccgaa
     1021 atgaaaaaga aattcaaaaa ctgcgaagtg cgttgcgacg aatctaacca ctgcgttgaa
     1081 gttcgctgtt ctgacaccaa atacaccctg tgctaatgac ggctgctaac aaagcccgaa
     1141 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1201 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     1261 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1321 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1381 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1441 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1501 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1561 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1621 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1681 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     1741 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1801 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     1861 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     1921 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     1981 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     2041 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     2101 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     2161 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     2221 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     2281 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     2341 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     2401 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     2461 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     2521 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     2581 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     2641 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     2701 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2761 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2821 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2881 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2941 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3001 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3061 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3121 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3181 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3241 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3301 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3361 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3421 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3481 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     3541 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     3601 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3661 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3721 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3781 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3841 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3901 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3961 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     4021 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     4081 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     4141 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     4201 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4261 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     4321 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4381 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4441 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4501 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4561 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4621 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4681 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4741 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4801 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4861 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4921 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4981 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5041 gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5101 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5161 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5221 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5281 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5341 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5401 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5461 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5521 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5581 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5641 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5701 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5761 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5821 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5881 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5941 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6001 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
//
