LOCUS       sequence-204361-        6026 bp    DNA     circular SYN 14-APR-2025
DEFINITION  plasmid for expression of WT colicin endonuclease and immunity pair 
            2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6026)
  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 6026)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-colEwt2/Imwt2
FEATURES             Location/Qualifiers
     source          1..6026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      51..639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     540..559
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     793..810
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    825..964
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(890..909)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1050..1072
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1066..1257)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3109..3128
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3148..3225)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3431..3450
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3534..3553
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3534..3552
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3553..3577
                     /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             3592..3614
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4292..4309
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4362..4380)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4376..4423
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4460..4915
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4547..4566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4757..4778
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4941..5045
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5046..5114,5115..5906)
                     /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(5264..5283)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
       61 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      121 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      181 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
      241 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
      301 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
      361 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
      421 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
      481 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
      541 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
      601 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
      661 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
      721 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
      781 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
      841 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
      901 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
      961 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     1021 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     1081 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     1141 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     1201 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     1261 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     1321 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     1381 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     1441 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     1501 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     1561 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     1621 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     1681 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     1741 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     1801 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     1861 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     1921 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     1981 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     2041 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa actgtcgtgc
     2101 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     2161 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     2221 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     2281 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     2341 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     2401 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     2461 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     2521 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     2581 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     2641 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     2701 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     2761 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     2821 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     2881 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     2941 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     3001 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     3061 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     3121 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     3181 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     3241 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     3301 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     3361 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     3421 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     3481 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     3541 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     3601 ctttaagaag gagatatacc atggagagta aacggaataa gccagggaag gcgacaggta
     3661 aaggtaaacc agttggtgat aaatggctgg atgatgcagg taaagattca ggagcgccaa
     3721 ttccagatcg cattgctgat aagttgcgtg ataaagaatt taaaaacttt gacgatttcc
     3781 ggaagaaatt ctgggaagaa gtgtcaaaag atcccgatct tagtaagcaa tttaaaggca
     3841 gtaataagac gaacattcaa aagggaaaag caccttttgc aaggaagaaa gaccaagtag
     3901 gtggtaggga acgctttgaa ttacatcatg ataaaccaat cagtcaggat ggtggtgtct
     3961 atgatatgaa taatatcaga gtgaccacac ctaagcgaca tattgatatt catcggggta
     4021 agtaaaaatg gaactgaaac atagtattag tgattatacc gaggctgaat ttctggagtt
     4081 tgtaaaaaaa atatgtagag ctgaaggtgc tactgaagag gatgacaata aattagtgag
     4141 agagtttgag cgattaactg agcacccaga cggttcagat ctgatttatt atcctcgcga
     4201 tgacagggaa gatagtcctg aagggattgt caaggaaatt aaagaatggc gagctgctaa
     4261 cggtaagtca ggatttaaac agggcctcga gcaccaccac caccaccact gagatccggc
     4321 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     4381 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     4441 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4501 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4561 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4621 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     4681 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4741 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4801 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4861 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     4921 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4981 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5041 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5101 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5161 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5221 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5281 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5341 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5401 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5461 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5521 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5581 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5641 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5701 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5761 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5821 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5881 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5941 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6001 tcatgaccaa aatcccttaa cgtgag
//
