LOCUS       sequence-433794-        7442 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Ec86 operon (effector, ncRNA and reverse transcriptase) in
            Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7442)
  AUTHORS   Wang Y, Guan Z, Wang C, Nie Y, Chen Y, Qian Z, Cui Y, Xu H, Wang Q, 
            Zhao F, Zhang D, Tao P, Sun M, Yin P, Jin S, Wu S, Zou T
  TITLE     Cryo-EM structures of Escherichia coli Ec86 retron complexes reveal 
            architecture and defence mechanism.
  JOURNAL   Nat Microbiol. 2022 Sep;7(9):1480-1489. doi: 
            10.1038/s41564-022-01197-7. Epub 2022 Aug 18.
  PUBMED    35982312
REFERENCE   2  (bases 1 to 7442)
  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: 
            "10.1038/s41564-022-01197-7"; journalName: "Nat Microbiol"; date: 
            "2022-09"; volume: "7"; issue: "9"; pages: "1480-1489"
COMMENT     Sequence Label: pET21b-effector-ncRNA-rrt-His8
FEATURES             Location/Qualifiers
     source          1..7442
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(167..190)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             2250..2272
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2287..2311
                     /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)."
     primer_bind     complement(2311..2330)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2312..2330)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2414..2433)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2639..2716
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2717..3799
                     /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     complement(2736..2755)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4608..4799
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4793..4815)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4901..5040
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4956..4975
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5055..5072)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5226..5814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5306..5325)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5985..6776,6777..6845))
                     /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     6608..6627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6846..6950)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6976..7431)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7113..7134)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7325..7344
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gactcagtga tggtgatggt
      181 gatggtgatg ctcgagggtc ttcgctttat ttaaagggtt ctttccatat tttttttcta
      241 atttgctaat ataagttatt aatctcctat ggctttttga atccacactt aaaataaatg
      301 acaaccatcc cctaacgtgt tctatctcag aagacttacc gcaaaatata tgatgtatct
      361 ttgctctaat ttctttatat ttttctctac ctatcccaac tttctcttgt gaaataacta
      421 aacctgtaac tttcctctga ctacgaggcc cactaataca agtttttttt gagttaataa
      481 ccaatccttc acttgggatt atagaaaata aaaaatcacg tgctttaaca acctttttca
      541 tagactgtgc agataaggtg agatcatcgg catatctcgt atatatcaag ccccgactac
      601 ctgcataacc ctgaatacga taatcaagtt tagaacatat tagattagct aatttaggtg
      661 atgatggagc accttgtggt agcagatttt tataacaaca tatttttgtc aaaactgaag
      721 atattagtcg attataacca agagaatgga acactccaaa aactttgtta gcagttaaac
      781 ttgggaaaaa atcctccaaa tcaatattca gtataaagtt tgccccaata tgcggggtag
      841 cattattcaa aatagattgg tgcttttcaa atccaataga aaaaggagat gacgacagtt
      901 tatctaaaat gttacgtaga acccatcctt gtaaggcttt aagttctcga gaaggttggt
      961 aaatggttct cattctcttc tctgggcctt tcttttctac agtgtagatc ctatagcgaa
     1021 aatcagctgt atagattaac aaccgaagtg tttcaacaga tatgcgagtc gccttagaca
     1081 tgtcatgcaa attgttcatg acaggtaggc cgagatttct caatctaaaa gtgttcaaat
     1141 attcagcgga tttcatgaaa gttgcgcacc cttacgtcag aaaaaacggg tttcctggtt
     1201 ggctcggaga gcatcaggcg atgctctccg ttccaacaag gaaaacagac agtaactcag
     1261 attcaatgca ggatgccgaa acaacatcca gaggttgacc ttaatgataa acctctcgct
     1321 aagggtgcgc atacggaatc ttatcatagt taaatgttga ttttctacgg ttttcaaaat
     1381 tcataatctc aagccgcaat cggtcaagtg tctttctatc aaagacgctc ctaacatagc
     1441 tagcccccaa agcagtgacc tgataaccat cagtcatttg aagtattttt cgttcattta
     1501 ttagacgaga aacaacggat ctaacaataa ttttagataa aacatcatct tgctttatcg
     1561 ctttaaaagc tagttcacac agtgtgcggt agttgacact atccagtaaa tagatacaag
     1621 gcaatagaaa ccgctctgcg tataatatat ttccaatatc ttttgatact ttattttctt
     1681 tcctaacctt cttaatagat gccattaatt ttttatataa tcgtaatttt ctggcaacat
     1741 caatagatga atcacacatt tcttttagtt catttgaact acaacgcaaa acagattttg
     1801 aattaaactt acgcaacagg cgaacaggac catagttaat aaagctacgt tttgatttaa
     1861 attttgcatc ttgaatgcaa atcaactttc tccttaagtt ttcattatta gagaacgcac
     1921 caagctctgt gaaagacccc ggactttcag gaaataaaat tattacatcg acagcttcag
     1981 ccagaatatt ttctaaactt aaaaggctat gctgcccttg accagccaaa agatcatcaa
     2041 atagatcttc tggataaaat atatcaacat cactgaactt tgctaaaaat tgtgataatt
     2101 gatgacgcca agattgatga ttagcaacgt caccaccaca tagaaaaatg gttattttaa
     2161 tattagctcc tcgatagaag cctttctttg tgagatgtcc tataagctgt tgttgctgct
     2221 catcggtaaa ttttttattc atatgtatat ctccttctta aagttaaaca aaattatttc
     2281 tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg
     2341 atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     2401 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
     2461 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg
     2521 ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac
     2581 ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga
     2641 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     2701 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     2761 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     2821 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     2881 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     2941 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     3001 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     3061 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     3121 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     3181 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     3241 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     3301 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     3361 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     3421 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     3481 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     3541 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     3601 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     3661 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     3721 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     3781 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     3841 cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
     3901 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     3961 aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga
     4021 tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca
     4081 ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc
     4141 cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc
     4201 ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca
     4261 aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc
     4321 tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct
     4381 gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag
     4441 tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca
     4501 gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg
     4561 gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg
     4621 aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga
     4681 aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg
     4741 ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     4801 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     4861 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     4921 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     4981 agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     5041 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5101 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5161 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5221 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5281 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5341 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5401 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5461 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5521 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5581 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5641 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     5701 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5761 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5821 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5881 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5941 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6001 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6061 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6121 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6181 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6241 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6301 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6361 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6421 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6481 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6541 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6601 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6661 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6721 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6781 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6841 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6901 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6961 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     7021 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     7081 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     7141 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     7201 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     7261 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     7321 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     7381 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     7441 ca
//
