LOCUS       sequence-213237-        8312 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ala-tRNA synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8312)
  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 8312)
  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: B4-1
FEATURES             Location/Qualifiers
     source          1..8312
                     /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(3086..3104)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3100..3147
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3322..3327,3328..3344,3345..3350))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3377..3395)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3463..3567
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3568..3636,3637..4428)
                     /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(3786..3805)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4599..5187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5088..5107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5341..5358
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5373..5512
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5438..5457)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5598..5620
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5614..5805)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7117..8199)
                     /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     8161..8180
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8200..8277)
                     /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 tttaagcaag agcaccgctg agatccgtca ggcgtttctc
      421 gactttttcc atagtaaggg acatcaggta gttgccagca gctccctggt accccataac
      481 gacccaactt tgttgtttac caacgccggg atgaaccagt tcaaggatgt gttccttggg
      541 ctcgacaagc gtaattattc ccgcgctacc acttcccaac gctgcgtgcg tgcgggtggt
      601 aaacacaacg acctggaaaa cgtcggttac accgcgcgtc accatacctt cttcgaaatg
      661 ctgggcaact tcagcttcgg cgactatttc aaacacgatg ccattcagtt tgcatgggaa
      721 ctgctgacca gcgaaaaatg gtttgccctg ccgaaagagc gtctgtgggt taccgtctat
      781 gaaagcgacg acgaagccta cgaaatctgg gaaaaagaag tagggatccc gcgcgaacgt
      841 attattcgca tcggcgataa caaaggtgcg ccatacgcat ctgacaactt ctggcagatg
      901 ggtgacactg gtccgtgcgg cccgtgcacc gaaatcttct acgatcacgg cgaccacatt
      961 tgggggggcc ctccgggaag cccggaagaa gacggcgacc gctacattga gatctggaac
     1021 atcgtcttca tgcagttcaa ccgccaggcc gatggcacga tggaaccgct gccgaagccg
     1081 tctgtagata ccggtatggg tctggagcgt attgctgcgg tgctgcaaca cgttaactct
     1141 aactatgaca tcgacctgtt ccgcacgctg atccaggcgg tagcgaaagt cactggcgca
     1201 accgatctga gcaataaatc gctgcgcgta atcgctgacc acattcgttc ttgtgcgttc
     1261 ctgatcgcgg atggcgtaat gccgtccaat gaaaaccgtg gttatgtact gcgtcgtatc
     1321 attcgtcgcg cagtgcgtca cggtaatatg ctcggcgcga aagaaacctt cttctacaaa
     1381 ctggttggtc cgctgatcga cgttatgggc tctgcgggtg aagacctgaa acgccagcag
     1441 gcgcaggttg agcaggtgct gaagactgaa gaagagcagt ttgctcgtac tctggagcgc
     1501 ggtctggcgt tgctggatga agagctggca aaactttctg gtgatacgct ggatggtgaa
     1561 actgctttcc gtctgtacga cacctatggc ttcccggttg acctgacggc tgatgtttgt
     1621 cgtgagcgca acatcaaagt tgacgaagct ggttttgaag ctgcaatgga agagcagcgt
     1681 cgtcgcgcgc gcgaagccag cggctttggt gccgattaca acgcaatgat ccgtgttgac
     1741 agtgcatctg aatttaaagg ctatgaccat ctggaactga acggcaaagt gactgcgctg
     1801 tttgttgatg gtaaagcggt tgatgccatc aatgcaggcc aggaagctgt ggtcgtgctg
     1861 gatcaaacgc cattctatgc ggaatccggc ggtcaggttg gcgataaagg cgaactgaaa
     1921 ggcgctaact tctcctttgc ggtggaagat acgcagaaat acggccaggc gattggtcac
     1981 atcggtaaac ttgctgcggg ttctctgaaa gtgggcgacg cggtgcaggc tgatgttgat
     2041 gaggctcgtc gcgcccgtat tcgtctgaat cactccgcaa cgcacctgat gcacgctgcg
     2101 ctgcgccagg ttctgggtac tcatgtatcg cagaaaggtt cactggttaa cgacaaggtg
     2161 ctgcgcttcg acttctcaca caacgaagcg atgaaaccag aagagattcg tgcggtcgaa
     2221 gacctggtga acacacagat tcgtcgcaat ttgccgatcg aaaccaacat catggatctc
     2281 gaagcggcga aagcgaaagg tgcgatggcg ctgttcggcg agaagtatga tgagcgcgta
     2341 cgcgtgctga gcatgggcga tttctctacc gagttgtgtg gcggtactca cgccagccgc
     2401 actggtgata ttggtctgtt ccgcatcatc tctgaatcgg gtactgctgc aggcgttcgt
     2461 cgtatcgaag cggtaaccgg agaaggtgct atcgccaccg ttcatgcaga cagcgatcgc
     2521 ttaagcgaag tcgcgcatct gctgaaaggc gatagcaata atctggctga taaagtgcgc
     2581 tcagtactgg aacgtacgcg tcagctggaa aaagagttac aacagcttaa agaacaagct
     2641 gccgcacagg agagcgcaaa tctttccagt aaggcaattg atgttaatgg tgttaagctg
     2701 ttggttagcg agcttagcgg tgttgagccg aaaatgttgc gtaccatggt tgacgattta
     2761 aaaaatcagc tggggtcgac aattatcgtg ctggcaacgg tagtcgaagg taaggtttct
     2821 ctgattgcag gcgtatctaa ggacgtcaca gatcgtgtga aagcagggga actgattggt
     2881 atggtcgctc agcaggtggg cggcaagggt ggtggacgtc ctgacatggc gcaagccggt
     2941 ggtacggatg ctgcggcctt acctgcagcg ttagccagtg tgaaaggctg ggtcagcgcg
     3001 aaattgcaat attaaagcag aggccatatg ctcgaggatc cggctgctaa caaagcccga
     3061 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     3121 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca
     3181 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc
     3241 cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat
     3301 ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat
     3361 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     3421 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3481 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3541 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3601 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3661 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3721 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     3781 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     3841 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3901 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3961 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4021 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4081 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     4141 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4201 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4261 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4321 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4381 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4441 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4501 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4561 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4621 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4681 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4741 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4801 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4861 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4921 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4981 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5041 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5101 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5161 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5221 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5281 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5341 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     5401 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     5461 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     5521 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5581 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5641 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     5701 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     5761 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     5821 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     5881 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     5941 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     6001 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     6061 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     6121 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     6181 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     6241 caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg
     6301 ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg
     6361 cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt
     6421 agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc
     6481 aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat
     6541 gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg
     6601 ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg
     6661 gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg
     6721 gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc
     6781 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     6841 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     6901 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     6961 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     7021 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     7081 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     7141 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     7201 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     7261 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     7321 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     7381 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     7441 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     7501 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     7561 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     7621 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     7681 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     7741 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     7801 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     7861 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     7921 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     7981 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     8041 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     8101 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     8161 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     8221 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     8281 gatctcgacg ctctccctta tgcgactcct gc
//
