LOCUS       sequence-382969-        4684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of peptides in E. coli as GB1 fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4684)
  AUTHORS   Pantelejevs T, Zuazua-Villar P, Koczy O, Counsell AJ, Walsh SJ, 
            Robertson NS, Spring DR, Downs JA, Hyvonen M
  TITLE     A recombinant approach for stapled peptide discovery yields 
            inhibitors of the RAD51 recombinase.
  JOURNAL   Chem Sci. 2023 Nov 21;14(47):13915-13923. doi: 10.1039/d3sc03331g. 
            eCollection 2023 Dec 6.
  PUBMED    38075664
REFERENCE   2  (bases 1 to 4684)
  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: 
            "10.1039/d3sc03331g"; journalName: "Chem Sci"; date: "2023-11-21- 
            21"; volume: "14"; issue: "47"; pages: "13915-13923"
COMMENT     Sequence Label: pPEPT1
FEATURES             Location/Qualifiers
     source          1..4684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    15..39
                     /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             52..74
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             82..108
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             121..144
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             163..324
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             334..354
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             397..420
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     promoter        complement(434..452)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(435..452)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(510..528)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      524..571
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(807..828)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(978..2060)
                     /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     2022..2041
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2061..2138)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     2277..2296
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2450..2469)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2569..2591
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2629..2647)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2715..2819
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2820..2888,2889..3680)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3038..3057)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3851..4439
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4340..4359
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4593..4610
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(4681..4684,1..16)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4681..4684,1..15)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 acgactcact atagggaatt gtgagcggat aacaattcct ctagaaataa ttttgtttaa
       61 ctttaagaag gagatatatc catgtatcga ttaaataagg aggaataaac catgtctgct
      121 tggagccacc cgcagttcga gaaatctggc actagtggca cctacaaact gatcctgaac
      181 ggtaaaaccc tgaaaggtga aaccaccacc gaagctgtag acgctgctac tgctgaaaaa
      241 gttttcaaac agtacgctaa cgacaacggt gtggacggtg aatggaccta cgacgacgct
      301 accaaaacct tcacggttac ggaaagcggt accgaaaacc tgtacttcca gggatccgac
      361 cctggagacc cgaaggtctc tgaccctggc tcgagccatc accatcacca tcaccatcac
      421 taagcttgag tattctatag tgtcacctaa atcccagctt gatccggctg ctaacaaagc
      481 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
      541 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggataacc
      601 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
      661 ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
      721 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
      781 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
      841 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca cagcttgcat
      901 gcctgcaggt cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactta
      961 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     1021 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     1081 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     1141 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     1201 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     1261 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     1321 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     1381 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     1441 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     1501 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     1561 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     1621 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     1681 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     1741 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     1801 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     1861 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     1921 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     1981 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     2041 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     2101 gccataccgc gaaaggtttt gcaccattcg atggtgtcgg cgagctcgaa ttgtgaacca
     2161 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     2221 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     2281 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     2341 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcctgatg cggtattttc
     2401 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     2461 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     2521 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     2581 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     2641 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     2701 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2761 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2821 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2881 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2941 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3001 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3061 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3121 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3181 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3241 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     3301 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     3361 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3421 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3481 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttctc
     3541 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3601 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3661 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3721 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3781 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3841 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3901 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3961 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4021 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4081 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4141 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4201 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4261 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4321 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4381 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4441 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4501 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4561 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4621 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgat
     4681 taat
//
