LOCUS       sequence-231790-        8470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  translational release factor 3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8470)
  AUTHORS   Villarreal F, Contreras-Llano LE, Chavez M, Ding Y, Fan J, Pan T, 
            Tan C
  TITLE     Synthetic microbial consortia enable rapid assembly of pure 
            translation machinery.
  JOURNAL   Nat Chem Biol. 2018 Jan;14(1):29-35. doi: 10.1038/nchembio.2514. 
            Epub 2017 Nov 13.
  PUBMED    29131146
REFERENCE   2  (bases 1 to 8470)
  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/nchembio.2514"; journalName: "Nat Chem Biol"; date: 
            "2018-01"; volume: "14"; issue: "1"; pages: "29-35"
COMMENT     Sequence Label: B1-3
FEATURES             Location/Qualifiers
     source          1..8470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             91..108
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1772..1790)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1786..1833
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2008..2013,2014..2030,2031..2036))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2063..2081)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2149..2253
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2254..2322,2323..3114)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2472..2491)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4484..5072
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4973..4992
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5226..5243
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5258..5397
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5323..5342)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5483..5505
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5499..5690)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7002..8084)
                     /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     8046..8065
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8085..8162)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8368..8387
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgagag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg catcatcatc accatcattt aacgttgtct
      121 ccttatttgc aagaggtggc gaagcgccgc acttttgcca ttatttctca cccggacgcc
      181 ggtaagacta ccatcaccga gaaggtgctg ctgttcggac aggccattca gaccgccggt
      241 acagtaaaag gccgtggttc caaccagcac gctaagtcgg actggatgga gatggaaaag
      301 cagcgtggga tctccattac tacgtctgtg atgcagtttc cgtatcacga ttgcctggtt
      361 aacctgctcg acaccccggg gcacgaagac ttctcggaag atacctatcg taccctgacg
      421 gcggtggact gctgcctgat ggttatcgac gccgcaaaag gtgttgaaga tcgtacccgt
      481 aagctgatgg aagttacccg tctgcgcgac acgccgatcc tcacctttat gaacaaactt
      541 gaccgtgata tccgcgaccc gatggagctg ctcgatgaag ttgagaacga gctgaaaatc
      601 ggctgtgcgc cgatcacctg gccgattggc tgcggcaagc tgtttaaagg cgtttaccac
      661 ctttataaag acgaaaccta tctctatcag agcggtaaag gccacaccat tcaggaagtc
      721 cgcattgtta aagggctgaa taacccggat ctcgatgctg cggttggtga agatctggca
      781 cagcagctgc gtgacgaact ggaactggtg aaaggcgcgt ctaacgagtt cgacaaagag
      841 ctgttccttg cgggcgaaat cactccggta ttcttcggta ctgcgctggg taacttcggc
      901 gtcgatcata tgttggatgg cctggtggag tgggcacctg cgccgatgcc gcgtcagact
      961 gatacccgta ccgtagaagc gagcgaagat aaatttaccg gcttcgtatt taaaattcag
     1021 gccaacatgg acccgaaaca ccgcgaccgc gtggcgttta tgcgtgtggt gtccggtaaa
     1081 tatgaaaaag gcatgaaact gcgccaggtg cgcactgcga aagatgtggt gatctccgac
     1141 gcgctgacct ttatggcggg tgaccgttcg cacgttgaag aagcgtatcc gggcgatatc
     1201 ctcggcctgc acaaccacgg caccattcag atcggcgaca cctttaccca gggtgagatg
     1261 atgaagttca ccggtattcc gaacttcgca ccagaactgt tccgtcgtat ccgcctgaaa
     1321 gatccgctga agcaaaaaca gctgctcaaa gggctggtac agctttccga agagggcgcg
     1381 gtgcaggtgt tccgtccaat ctccaacaac gatctgatcg ttggtgcagt tggtgtgctg
     1441 cagtttgatg tggtggtagc gcgcctgaag agcgaataca acgttgaagc agtgtatgag
     1501 tcagtcaacg ttgccactgc ccgctgggta gaatgtgcag acgcgaagaa attcgaagag
     1561 ttcaagcgta agaacgaaag ccaactggcg cttgatggcg gcgataacct cgcttacatc
     1621 gctaccagca tggtcaacct gcgcctggca caggaacgtt atccggacgt tcagttccac
     1681 cagacccgcg agcattatta aagcagcggc catatgctcg aggatccggc tgctaacaaa
     1741 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1801 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat
     1861 cccgcaagag gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga
     1921 cggtgccgag gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt
     1981 agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat
     2041 gagaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     2101 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     2161 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2221 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2281 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2341 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2401 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2461 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     2521 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2581 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2641 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2701 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2761 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     2821 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2881 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2941 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3001 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3061 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3121 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3181 atctagggaa ggtgcgaata agcggggaaa ttcttctcgg ctgactcagt catttcattt
     3241 cttcatgttt gagccgattt tttctcccgt aaatgccttg aatcagccta tttagaccgt
     3301 ttcttcgcca tttaaggcgt tatccccagt ttttagtgag atctctccca ctgacgtatc
     3361 atttggtccg cccgaaacag gttggccagc gtgaataaca tcgccagttg gttatcgttt
     3421 ttcagcaacc ccttgtatct ggctttcacg aagccgaact gtcgcttgat gatgcgaaat
     3481 gggtgctcca ccctggcccg gatgctggct ttcatgtatt cgatgttgat ggccgttttg
     3541 ttcttgcgtg gatgctgttt caaggttctt accttgccgg ggcgctcggc gatcagccag
     3601 tccacatcca cctcggccag ctcctcgcgc tgtggcgccc cttggtagcc ggcatcggct
     3661 gagacaaatt gctcctctcc atgcagcaga ttacccagct gattgaggtc atgctcgttg
     3721 gccgcggtgg tgaccaggct gtgggtcagg ccactcttgg catcgacacc aatgtgggcc
     3781 ttcatgccaa agtgccactg attgcctttc ttggtctgat gcatctccgg atcgcgttgc
     3841 tgctctttgt tcttggtcga gctgggtgcc tcaatgatgg tggcatcgac caaggtgcct
     3901 tgagtcatca tgacgcctgc ttcggccagc cagcgattga tggtcttgaa caattggcgg
     3961 gccagttgat gctgctccag caggtggcgg aaattcatga tggtggtgcg gtccggcaag
     4021 gcgctatcca gggataaccg ggcaaacaga cgcatggagg cgatttcgta cagagcatct
     4081 tccatcgcgc catcgctcag gttgtaccaa tgctgcatgc agtgaatgcg tagcatggtt
     4141 tccagcggat aaggtcgccg gccattacca gccttggggt aaaacggctc gatgacttcc
     4201 accatgtttt gccatggcag aatctgctcc atgcgggaca agaaaatctc ttttctggtc
     4261 tgacggcgct tactgctgaa ttcactgtcg gcgaaggtaa gttgatgact catgatgaac
     4321 cctgttctat ggctccagat gacaaacatg atctcatatc agggacttgt tcgcaccttc
     4381 cctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4441 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4501 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4561 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4621 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4681 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4741 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4801 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4861 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4921 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4981 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5041 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5101 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5161 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5221 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     5281 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     5341 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     5401 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5461 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5521 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     5581 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     5641 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     5701 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     5761 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     5821 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     5881 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     5941 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     6001 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     6061 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     6121 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     6181 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
     6241 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
     6301 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
     6361 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
     6421 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag
     6481 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
     6541 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
     6601 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
     6661 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     6721 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     6781 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     6841 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     6901 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     6961 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     7021 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     7081 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     7141 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     7201 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     7261 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     7321 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     7381 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     7441 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     7501 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     7561 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     7621 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     7681 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     7741 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     7801 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     7861 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     7921 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     7981 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     8041 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     8101 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     8161 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     8221 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     8281 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     8341 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     8401 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     8461 cccgcgaaat
//
