LOCUS       sequence-386236-        6356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Ec86 reverse transcriptase  in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6356)
  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 6356)
  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-rrt-His8
FEATURES             Location/Qualifiers
     source          1..6356
                     /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             1164..1186
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1201..1225
                     /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(1225..1244)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1226..1244)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1328..1347)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1553..1630
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1631..2713
                     /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(1650..1669)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3522..3713
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3707..3729)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3815..3954
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3870..3889
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3969..3986)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4140..4728)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4220..4239)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4899..5690,5691..5759))
                     /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     5522..5541
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5760..5864)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5890..6345)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6027..6048)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6239..6258
                     /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 tttcatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg
     1201 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
     1261 atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
     1321 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
     1381 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
     1441 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
     1501 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat
     1561 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     1621 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     1681 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     1741 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     1801 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     1861 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     1921 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     1981 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     2041 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     2101 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     2161 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     2221 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     2281 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     2341 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     2401 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     2461 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     2521 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     2581 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     2641 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2701 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat
     2761 ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     2821 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     2881 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     2941 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     3001 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg
     3061 tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg
     3121 gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt
     3181 ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa
     3241 cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     3301 taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt
     3361 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc
     3421 gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca
     3481 ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa
     3541 ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
     3601 acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg
     3661 agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     3721 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     3781 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     3841 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     3901 ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     3961 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4021 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4081 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4141 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4201 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4261 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4321 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4381 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4441 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4501 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4561 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4621 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4681 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4741 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4801 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4861 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4921 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4981 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5041 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5101 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5161 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc
     5221 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5281 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5341 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5401 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5461 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5521 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5581 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5641 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5701 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5761 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5821 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5881 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     5941 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     6001 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     6061 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     6121 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     6181 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     6241 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     6301 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
