LOCUS       sequence-213173-        6764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  translational release factor 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6764)
  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 6764)
  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: B4-4
FEATURES             Location/Qualifiers
     source          1..6764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     171..190
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     274..293
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        274..292
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             332..354
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             364..381
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1538..1556)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1552..1599
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1774..1779,1780..1796,1797..1802))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1829..1847)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1915..2019
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2020..2088,2089..2880)
                     /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(2238..2257)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3051..3639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3540..3559
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3793..3810
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3825..3964
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3890..3909)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4050..4072
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4066..4257)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5569..6651)
                     /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     6613..6632
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6652..6729)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
       61 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
      121 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
      181 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
      241 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattga
      301 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc
      361 atgcatcatc atcaccatca tttaaagcct tctatcgttg ccaaactgga agccctgcat
      421 gaacgccatg aagaagttca ggcgttgctg ggtgacgcgc aaactatcgc cgaccaggaa
      481 cgttttcgcg cattatcacg cgaatatgcg cagttaagtg atgtttcgcg ctgttttacc
      541 gactggcaac aggttcagga agatatcgaa accgcacaga tgatgctcga tgatcctgaa
      601 atgcgtgaga tggcgcagga tgaactgcgc gaagctaaag aaaaaagcga gcaactggaa
      661 cagcaattac aggttctgtt actgccaaaa gatcctgatg acgaacgtaa cgccttcctc
      721 gaagtccgag ccggaaccgg cggcgacgaa gcggcgctgt tcgcgggcga tctgttccgt
      781 atgtacagcc gttatgccga agcccgccgc tggcgggtag aaatcatgag cgccagcgag
      841 ggtgaacatg gtggttataa agagatcatc gccaaaatta gcggtgatgg tgtgtatggt
      901 cgtctgaaat ttgaatccgg cggtcatcgc gtgcaacgtg ttcctgctac ggaatcgcag
      961 ggtcgtattc atacttctgc ttgtaccgtt gcggtaatgc cagaactgcc tgacgcagaa
     1021 ctgccggaca tcaacccagc agatttacgc attgatactt tccgctcgtc aggggcgggt
     1081 ggtcagcacg ttaacaccac cgattcggca attcgtatta ctcacttgcc gaccgggatt
     1141 gttgttgaat gtcaggacga acgttcacaa cataaaaaca aagctaaagc actttctgtt
     1201 ctcggtgctc gcatccacgc tgctgaaatg gcaaaacgcc aacaggccga agcgtctacc
     1261 cgtcgtaacc tgctggggag tggcgatcgc agcgaccgta accgtactta caacttcccg
     1321 caggggcgcg ttaccgatca ccgcatcaac ctgacgctct accgcctgga tgaagtgatg
     1381 gaaggtaagc tggatatgct gattgaaccg attatccagg aacatcaggc cgaccaactg
     1441 gcggcgttgt ccgagcagga atattaaagc agaggccata tgctcgagga tccggctgct
     1501 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1561 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1621 ggatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca
     1681 gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac
     1741 tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc
     1801 aaacatgaga attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     1861 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     1921 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1981 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2041 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2101 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2161 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2221 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     2281 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2341 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2401 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2461 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2521 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     2581 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     2641 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     2701 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     2761 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     2821 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     2881 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     2941 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3001 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3061 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3121 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3181 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3241 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3301 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3361 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3421 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3481 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3541 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3601 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3661 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3721 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3781 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3841 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     3901 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     3961 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4021 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4081 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     4141 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     4201 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     4261 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     4321 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     4381 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     4441 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     4501 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     4561 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     4621 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4681 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct
     4741 gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca
     4801 agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt
     4861 ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat
     4921 gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc
     4981 aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg
     5041 atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca
     5101 tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga
     5161 agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc
     5221 agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     5281 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     5341 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     5401 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     5461 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     5521 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     5581 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5641 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     5701 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     5761 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     5821 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     5881 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     5941 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     6001 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6061 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6121 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6181 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6241 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6301 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6361 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     6421 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     6481 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     6541 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     6601 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     6661 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     6721 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgc
//
