LOCUS       sequence-204345-        6035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid for expression of computationally designed colicin 
            endonuclease and immunity pair, des4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6035)
  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 6035)
  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-colEdes4/Imdes4
FEATURES             Location/Qualifiers
     source          1..6035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      32..620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     521..540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     774..791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    806..945
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(871..890)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1031..1053
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1047..1238)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3090..3109
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3129..3206)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3412..3431
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3515..3534
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3515..3533
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3534..3558
                     /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             3573..3595
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4282..4299
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4352..4370)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4366..4413
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4450..4905
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4537..4556)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4747..4768
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4931..5035
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5036..5104,5105..5896)
                     /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(5254..5273)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
       61 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
      121 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
      181 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
      241 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
      301 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
      361 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
      421 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      481 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      541 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      601 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      661 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      721 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      781 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
      841 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
      901 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
      961 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     1021 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     1081 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     1141 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     1201 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     1261 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     1321 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     1381 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     1441 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     1501 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     1561 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     1621 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     1681 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     1741 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     1801 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     1861 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     1921 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     1981 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     2041 ttgcgctcac tgcccgcttt ccagtcggga aactgtcgtg ccagctgcat taatgaatcg
     2101 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     2161 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     2221 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     2281 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     2341 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     2401 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     2461 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     2521 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     2581 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     2641 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     2701 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     2761 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     2821 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     2881 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     2941 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     3001 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     3061 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     3121 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     3181 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     3241 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     3301 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     3361 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     3421 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     3481 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     3541 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     3601 catggagagt aaacggaata agccagggaa ggcgacaggt aaaggtaaac cagttggtga
     3661 taaatggctg gatgatgcag gtaaagattc aggagcgcca attccagatc gcattgctga
     3721 taagttgcgt gataaagaat ttaaaaactt tgacgatttc cggaagaaat tctgggaaga
     3781 agtgtcaaaa gatcccgatc ttagtaagca atttaaaggc gctaataagg atagtattag
     3841 taagggacaa gcaccttttg caaggaagaa agaccaagta ggtggtagga agacctttga
     3901 attacatcat gataaaccaa tcagtcagga tggtggtgtc tatgatatga ataatatcag
     3961 agtgaccaca cctaagcgac atattgatat tcatcggggt aagtaaaaat ggaactgaaa
     4021 catagtatta gtgattatac cgaggctgaa tttctggagt ttgtaaaaaa aatattacag
     4081 gcttcatcag gaggaggtgg taacgaggac ttaagtcgaa aattagtgga ggagtttgag
     4141 cgattaactg agcacccaga cggttcagat ctgatttatt atcctcgcga tgacagggaa
     4201 gatagtcctg aagggattgt caaggaaatt aaagaatggc gagctgctaa cggtaagtca
     4261 ggatttaaac agggcctcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     4321 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     4381 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     4441 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4501 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4561 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4621 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4681 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4741 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     4801 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     4861 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     4921 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4981 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5041 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5101 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5161 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5221 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5281 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5341 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5401 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5461 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5521 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     5581 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5641 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5701 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     5761 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5821 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5881 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5941 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6001 aatcccttaa cgtgagtttt cgttccactg agcgt
//
