LOCUS       sequence-204344-        6029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid for expression of computationally designed colicin 
            endonuclease and immunity pair, des3.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6029)
  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 6029)
  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-colEdes3.5/Imdes3.5
FEATURES             Location/Qualifiers
     source          1..6029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      15..603
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     504..523
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     757..774
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    789..928
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(854..873)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1014..1036
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1030..1221)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3073..3092
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3112..3189)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3395..3414
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3498..3517
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3498..3516
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3517..3541
                     /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             3556..3578
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4259..4276
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4329..4347)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4343..4390
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4427..4882
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4514..4533)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4724..4745
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4908..5012
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5013..5081,5082..5873)
                     /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(5231..5250)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
       61 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      121 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
      181 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
      241 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
      301 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
      361 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
      421 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
      481 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      541 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      601 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
      661 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
      721 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      781 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
      841 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
      901 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
      961 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     1021 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     1081 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     1141 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     1201 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     1261 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     1321 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     1381 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     1441 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     1501 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     1561 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     1621 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     1681 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     1741 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     1801 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     1861 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     1921 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     1981 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     2041 tttccagtcg ggaaactgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     2101 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     2161 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     2221 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     2281 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     2341 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     2401 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     2461 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     2521 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     2581 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     2641 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     2701 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     2761 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     2821 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     2881 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     2941 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     3001 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     3061 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     3121 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     3181 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     3241 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     3301 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     3361 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     3421 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     3481 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     3541 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatggag agtaaacgga
     3601 ataagccagg gaaggcgaca ggtaaaggta aaccagttgg tgacaaatgg ctggacgatg
     3661 ctggaaagga tagcggcgca cccatccccg atcgtatcgc tgacaaatta cgcgataagg
     3721 agtttaaaaa cttcgacgac tttcgtaaga agttttggaa ggaagttgcc aaagatcccg
     3781 atttggccaa acagttctcg aaggcaaatc agcgcaacat taaggacggt acggcccctt
     3841 ttgcccgcga atccgatcaa gtggggggcc gtcatcagta tgaactgcac catgacaaac
     3901 caatctcaca ggacgggggc gtctatgata tgaataatat cagagtgacc acacctaagc
     3961 gacatattga tattcatcgg ggtaagtaaa aatggaactg aaacatagta ttagtgatta
     4021 taccgaggct gaatttctgg agtttgttaa ggacattttt cgtttgtccc gtccgcaaga
     4081 cacagactta caggtgaagc tggttcttga atttaagcgc ttaaccgagc acccagacgg
     4141 ctcggacttg atctattatc cccgctccga tcgtgaagac agtccagaag gcatcgtcaa
     4201 ggagattaaa gagtggcgag ctgctaacgg taagtcagga tttaaacagg gcctcgagca
     4261 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     4321 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     4381 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     4441 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4501 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4561 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4621 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     4681 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4741 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     4801 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4861 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4921 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4981 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5041 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5101 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5161 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5221 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5281 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5341 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5401 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5461 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5521 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     5581 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5641 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5701 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5761 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     5821 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5881 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5941 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6001 tccactgagc gtcagacccc gtagaaaag
//
