LOCUS       sequence-213213-        8311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Leu-tRNA synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8311)
  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 8311)
  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: B5-1
FEATURES             Location/Qualifiers
     source          1..8311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2879..2896
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3020..3038)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3034..3081
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3256..3261,3262..3278,3279..3284))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3311..3329)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3397..3501
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3502..3570,3571..4362)
                     /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(3720..3739)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4533..5121
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5022..5041
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5275..5292
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5307..5446
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5372..5391)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5532..5554
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5548..5739)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7051..8133)
                     /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     8095..8114
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8134..8211)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgagagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatgca
      301 tcaagagcaa taccgcccgg aagagaaaga atccaaagta cagcttcatt gggatgagaa
      361 gcgcacattt gaagtaaccg aagacgagag caaagagaag tattactgcc tgtctatgct
      421 tccctatcct tctggtcgac tacacatggg ccacgtacgt aactacacca tcggtgacgt
      481 gatcgcccgc taccagcgta tgctgggcaa aaacgtcctg cagccgatcg gctgggacgc
      541 gtttggtctg cctgcggaag gcgcggcggt gaaaaacaac accgctccgg caccgtggac
      601 gtacgacaac atcgcgtata tgaaaaacca gctcaaaatg ctgggctttg gttatgactg
      661 gagccgcgag ctggcaacct gtacgccgga atactaccgt tgggaacaga aattcttcac
      721 cgagctgtat aaaaaaggcc tggtatataa gaagacttct gcggtcaact ggtgcccgaa
      781 cgaccagacc gtactggcga acgaacaagt tatcgacggc tgctgctggc gctgcgatac
      841 caaagttgaa cgtaaagaga tcccgcagtg gtttatcaaa atcactgctt acgctgacga
      901 gctgctcaac gatctggata aactggatca ctggccagac accgttaaaa ccatgcagcg
      961 taactggatc ggtcgttccg aaggcgtgga gatcaccttc aacgttaacg actatgacaa
     1021 cacgctgacc gtttacacta cccgcccgga cacctttatg ggttgtacct acctggcggt
     1081 agctgcgggt catccgctgg cgcagaaagc ggcggaaaat aatcctgaac tggcggcctt
     1141 tattgacgaa tgccgtaaca ccaaagttgc cgaagctgaa atggcgacga tggagaaaaa
     1201 aggcgtcgat actggcttta aagcggttca cccattaacg ggcgaagaaa ttcccgtttg
     1261 ggcagcaaac ttcgtattga tggagtacgg cacgggcgca gttatggcgg taccggggca
     1321 cgaccagcgc gactacgagt ttgcctctaa atacggcctg aacatcaaac cggttatcct
     1381 ggcagctgac ggctctgagc cagatctttc tcagcaagcc ctgactgaaa aaggcgtgct
     1441 gttcaactct ggcgagttca acggtcttga ccatgaagcg gccttcaacg ccatcgccga
     1501 taaactgact gcgatgggcg ttggcgagcg taaagtgaac taccgcctgc gcgactgggg
     1561 tgtttcccgt cagcgttact ggggcgcgcc gattccgatg gtgacgctgg aagacggtac
     1621 cgtaatgccg accccggacg accagctgcc ggtgatcctg ccggaagatg tggtaatgga
     1681 cggcattacc agcccgatta aagcagatcc ggagtgggcg aaaactaccg ttaacggtat
     1741 gccagcactg cgtgaaaccg acactttcga cacctttatg gagtcctcct ggtactatgc
     1801 gcgctacact tgcccgcagt acaaagaagg tatgctggat tccgaagcgg ctaactactg
     1861 gctgccggtg gatatctaca ttggtggtat tgaacacgcc attatgcacc tgctctactt
     1921 ccgcttcttc cacaaactga tgcgtgatgc aggcatggtg aactctgacg aaccagcgaa
     1981 acagttgctg tgtcagggta tggtgctggc agatgccttc tactatgttg gcgaaaacgg
     2041 cgaacgtaac tgggtttccc cggttgatgc tatcgttgaa cgtgacgaga aaggccgtat
     2101 cgtgaaagcg aaagatgcgg caggccatga actggtttat accggcatga gcaaaatgtc
     2161 caagtcgaag aacaacggta tcgacccgca ggtgatggtt gaacgttacg gcgcggacac
     2221 cgttcgtctg tttatgatgt ttgcttctcc ggctgatatg actctcgaat ggcaggaatc
     2281 cggtgtggaa ggggctaacc gcttcctgaa acgtgtctgg aaactggttt acgagcacac
     2341 agcaaaaggt gatgttgcgg cactgaacgt tgatgcgctg actgaaaatc agaaagcgct
     2401 gcgtcgcgat gtgcataaaa cgatcgctaa agtgaccgat gatatcggcc gtcgtcagac
     2461 cttcaacacc gcaattgcgg cgattatgga gctgatgaac aaactggcga aagcaccaac
     2521 cgatggcgag caggatcgcg ctctgatgca ggaagcactg ctggccgttg tccgtatgct
     2581 taacccgttc accccgcaca tctgcttcac gctgtggcag gaactgaaag gcgaaggcga
     2641 tatcgacaac gcgccgtggc cggttgctga cgaaaaagcg atggtggaag actccacgct
     2701 ggtcgtggtg caggttaacg gtaaagtccg tgccaaaatc accgttccgg tggacgcaac
     2761 ggaagaacag gttcgcgaac gtgctggcca ggaacatctg gtagcaaaat atcttgatgg
     2821 cgttactgta cgtaaagtga tttacgtacc aggtaaactc ctcaatctgg tcgttggcca
     2881 tcatcatcac catcattaaa gcgttgtgcc tgtagccgac gtactccgcg taacgccagc
     2941 ttctattaaa gcagaggcca tatgctcgag gatccggctg ctaacaaagc ccgaaaggaa
     3001 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     3061 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc cgcaagaggc
     3121 ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga
     3181 tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact
     3241 gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga gaattcttga
     3301 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     3361 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     3421 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     3481 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     3541 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     3601 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     3661 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     3721 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     3781 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     3841 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3901 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3961 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     4021 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     4081 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     4141 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     4201 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     4261 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     4321 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     4381 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4441 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4501 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4561 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4621 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4681 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4741 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4801 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4861 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4921 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4981 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5041 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5101 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5161 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5221 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5281 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     5341 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5401 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     5461 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5521 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5581 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     5641 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     5701 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     5761 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     5821 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     5881 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     5941 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     6001 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     6061 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     6121 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     6181 caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt
     6241 gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt
     6301 cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag
     6361 gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg
     6421 gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc
     6481 gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt taggctggta
     6541 agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc
     6601 atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag
     6661 gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg
     6721 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     6781 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     6841 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     6901 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     6961 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     7021 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     7081 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     7141 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     7201 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     7261 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     7321 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     7381 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     7441 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     7501 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     7561 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     7621 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     7681 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     7741 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     7801 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     7861 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     7921 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     7981 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     8041 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     8101 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     8161 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     8221 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     8281 tgagcaccgc cgccgcaagg aatggtgcat g
//
