LOCUS       sequence-204610-        6020 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid for expression of computationally designed colicin 
            endonuclease and immunity pair, des8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6020)
  AUTHORS   Netzer R, Listov D, Lipsh R, Dym O, Albeck S, Knop O, Kleanthous C, 
            Fleishman SJ
  TITLE     Ultrahigh specificity in a network of computationally designed 
            protein-interaction pairs.
  JOURNAL   Nat Commun. 2018 Dec 11;9(1):5286. doi: 10.1038/s41467-018-07722-9.
  PUBMED    30538236
REFERENCE   2  (bases 1 to 6020)
  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: "Nat 
            Commun."; date: "2018-12-11"; volume: "9(1):5286. doi"; pages: " 
            10.1038/s41467-018-07722-9"
COMMENT     Sequence Label: pET21d-colEdes8/Imdes8
FEATURES             Location/Qualifiers
     source          1..6020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      22..610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     511..530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     764..781
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    796..935
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(861..880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1021..1043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1037..1228)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3080..3099
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3119..3196)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3402..3421
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3505..3524
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3505..3523
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3524..3548
                     /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             3563..3585
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4257..4274
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4327..4345)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4341..4388
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4425..4880
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4512..4531)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4722..4743
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4906..5010
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5011..5079,5080..5871)
                     /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     complement(5229..5248)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
       61 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
      121 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
      181 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
      241 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
      301 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
      361 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
      421 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
      481 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
      541 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
      601 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      661 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      721 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      781 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
      841 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
      901 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
      961 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     1021 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     1081 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     1141 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     1201 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     1261 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     1321 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     1381 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     1441 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     1501 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     1561 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     1621 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     1681 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     1741 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     1801 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     1861 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     1921 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     1981 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     2041 tgcccgcttt ccagtcggga aactgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     2101 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     2161 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     2221 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     2281 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     2341 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     2401 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     2461 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     2521 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     2581 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     2641 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     2701 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     2761 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     2821 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     2881 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     2941 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     3001 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     3061 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     3121 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     3181 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     3241 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     3301 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     3361 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     3421 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     3481 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
     3541 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac catggagagt
     3601 aaacggaata agccagggaa ggcgacaggt aaaggtaaac cagttggtga caagtggctg
     3661 gatgacgctg gtaaagactc tggtgctccg atcccggatc gtattgcgga caaactgcgt
     3721 gacaaagaat tcaaaaactt cgacgacttc cgtaaaaagt tctgggaaga agtttccaaa
     3781 gacccggacc tggcgaagca gttcaaccgt caaaacaaaa agaacatcaa acagggtcag
     3841 gcgccgttcg cgccacagaa agaccaggtt ggcggtcgta aaaccttcga actgcaccac
     3901 gacaaaccga tctctcagga cggtggtgtt tacgatatga acaacatcag agtgaccaca
     3961 cctaagcgac atattgatat tcatcggggt aagtaaaaat ggaactgaaa catagtatta
     4021 gtgattatac cgaggctgaa tttctggagt ttgtgaaaaa gatcgaaaac accaccgacg
     4081 aagaaaagca gaaagaactg gttgaagaat tcgaacgtct gaccgaacac ccgtctggtt
     4141 ctgacctggt ttactacccg cgtgacgacc gcgaggactc tccggaaggt atcgttaaag
     4201 aaatcaaaga atggcgagct gctaacggta agtcaggatt taaacagggc ctcgagcacc
     4261 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     4321 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     4381 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     4441 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4501 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4561 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4621 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4681 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4741 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4801 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4861 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4921 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4981 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5041 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5101 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5161 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5221 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5281 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5341 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5401 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5461 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5521 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     5581 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5641 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5701 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5761 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5821 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5881 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5941 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6001 cactgagcgt cagaccccgt
//
