LOCUS       sequence-231784-        8498 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ile-tRNA synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8498)
  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 8498)
  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: B2-1
FEATURES             Location/Qualifiers
     source          1..8498
                     /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(2999..3017)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3013..3060
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3235..3240,3241..3257,3258..3263))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3290..3308)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3376..3480
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3481..3549,3550..4341)
                     /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(3699..3718)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4512..5100
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5001..5020
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5254..5271
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5286..5425
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5351..5370)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5511..5533
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5527..5718)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7030..8112)
                     /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     8074..8093
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8113..8190)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8396..8415
                     /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 aagtgactat
      121 aaatcaaccc tgaatttgcc ggaaacaggg ttcccgatgc gtggcgatct cgccaagcgc
      181 gaacccggaa tgctggcgcg ttggactgat gatgatctgt acggcatcat ccgtgcggct
      241 aaaaaaggca aaaaaacctt cattctgcat gatggccctc cttatgcgaa tggcagcatt
      301 catattggtc actcggttaa caagattctg aaagacatta tcgtgaagtc caaagggctt
      361 tccggttatg actcgccgta tgtgcctggc tgggactgcc acggtctgcc gatcgagctg
      421 aaagtcgagc aagaatacgg taagccgggt gagaaattca ccgccgccga gttccgcgcc
      481 aagtgccgcg aatacgcggc gacccaggtt gacggtcaac gcaaagactt tatccgtctg
      541 ggcgtgctgg gcgactggtc gcacccgtac ctgaccatgg acttcaaaac tgaagccaac
      601 atcatccgcg cgctgggcaa aatcatcggc aacggtcacc tgcacaaagg cgcgaagcca
      661 gttcactggt gcgttgactg ccgttctgcg ctggcggaag cggaagttga gtattacgac
      721 aaaacttctc cgtccatcga cgttgctttc caggcagtcg atcaggatgc actgaaagca
      781 aaatttgccg taagcaacgt taacggccca atctcgctgg taatctggac caccacgccg
      841 tggactctgc ctgccaaccg cgcaatctct attgcaccag atttcgacta tgcgctggtg
      901 cagatcgacg gtcaggccgt gattctggcg aaagatctgg ttgaaagcgt aatgcagcgt
      961 atcggcgtga ccgattacac cattctcggc acggtaaaag gtgcggagct tgagctgctg
     1021 cgctttaccc atccgtttat gggcttcgac gttccggcaa tcctcggcga tcacgttacc
     1081 ctggatgccg gtaccggtgc cgttcacacc gcgcctggcc acggcccgga cgactatgtg
     1141 atcggtcaga aatacggcct ggaaaccgct aacccggttg gcccggacgg cacttatctg
     1201 ccgggcactt atccgacgct ggatggcgtg aacgtcttca aagcgaacga catcgtcgtt
     1261 gcgctgctgc aggaaaaagg cgcgctgctg cacgttgaga aaatgcagca cagctatccg
     1321 tgctgctggc gtcacaaaac gccgatcatc ttccgcgcga cgccgcagtg gttcgtcagc
     1381 atggatcaga aaggtctgcg tgcgcagtca ctgaaagaga tcaaaggcgt gcagtggatc
     1441 ccggactggg gccaggcgcg tatcgagtcg atggttgcta accgtcctga ctggtgtatc
     1501 tcccgtcagc gcacctgggg tgtaccgatg tcactgttcg tgcacaaaga cacggaagag
     1561 ctgcatccgc gtacccttga actgatggaa gaagtggcaa aacgcgttga agtcgatggc
     1621 atccaggcgt ggtgggatct cgatgcgaaa gagatcctcg gcgacgaagc tgatcagtac
     1681 gtgaaagtgc cggacacatt ggatgtatgg tttgactccg gatctaccca ctcttctgtt
     1741 gttgacgtgc gtccggaatt tgccggtcac gcagcggaca tgtatctgga aggttctgac
     1801 caacaccgcg gctggttcat gtcttcccta atgatctcca ccgcgatgaa gggtaaagcg
     1861 ccgtatcgtc aggtactgac ccacggcttt accgtggatg gtcagggccg caagatgtct
     1921 aaatccatcg gcaataccgt ttcgccgcag gatgtgatga acaaactggg cgcggatatt
     1981 ctgcgtctgt gggtggcatc aaccgactac accggtgaaa tggccgtttc tgacgagatc
     2041 ctgaaacgtg ctgccgatag ctatcgtcgt atccgtaaca ccgcgcgctt cctgctggca
     2101 aacctgaacg gttttgatcc agcaaaagat atggtgaaac cggaagagat ggtggtactg
     2161 gatcgctggg ccgtaggttg tgcgaaagcg gcacaggaag acatcctcaa ggcgtacgaa
     2221 gcatacgatt tccacgaagt ggtacagcgt ctgatgcgct tctgctccgt tgagatgggt
     2281 tccttctacc tcgacatcat caaagaccgt cagtacaccg ccaaagcgga cagtgtggcg
     2341 cgtcgtagct gccagactgc gctatatcac atcgcagaag cgctggtgcg ctggatggca
     2401 ccaatcctct ccttcaccgc tgatgaagtg tggggctacc tgccgggcga acgtgaaaaa
     2461 tacgtcttca ccggtgagtg gtacgaaggc ctgtttggcc tggcagacag tgaagcgatg
     2521 aacgatgcgt tctgggacga gctgttgaaa gtgcgtggcg aagtgaacaa agtcattgag
     2581 caagcgcgtg ccgacaagaa agtgggtggc tcgctggaag cggcagtaac cttgtatgca
     2641 gaaccggaac tgtcggcgaa actgaccgcg ctgggcgatg aattacgatt tgtcctgttg
     2701 acctccggcg ctaccgttgc agactataac gacgcacctg ctgatgctca gcagagcgaa
     2761 gtactcaaag ggctgaaagt cgcgttgagt aaagccgaag gtgagaagtg cccacgctgc
     2821 tggcactaca cccaggatgt cggcaaggtg gcggaacacg cagaaatctg cggccgctgt
     2881 gtcagcaacg tcgccggtga cggtgaaaaa cgtaagtttg cctattaaag cagcggccat
     2941 atgctcgagg atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     3001 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     3061 ctgaaaggag gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca
     3121 agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag
     3181 atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa
     3241 gcttatcgat gataagctgt caaacatgag aattcttgaa gacgaaaggg cctcgtgata
     3301 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     3361 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3421 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3481 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3541 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3601 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3661 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3721 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3781 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3841 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3901 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3961 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     4021 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4081 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4141 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4201 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4261 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4321 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4381 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4441 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4501 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4561 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4621 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4681 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4741 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4801 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4861 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4921 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4981 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5041 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5101 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5161 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     5221 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     5281 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     5341 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     5401 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     5461 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5521 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     5581 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     5641 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     5701 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     5761 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     5821 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     5881 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     5941 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     6001 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     6061 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     6121 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     6181 ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg agatggcgga
     6241 cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc gcaagaattg
     6301 attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc ttccattcag
     6361 gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg
     6421 gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc ataaatcgcc
     6481 gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag cgatccttga
     6541 agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa cgcgggcatc
     6601 ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc tcgcgtcgcg
     6661 aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc
     6721 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     6781 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     6841 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     6901 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     6961 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     7021 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     7081 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     7141 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     7201 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     7261 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     7321 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     7381 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     7441 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     7501 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     7561 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     7621 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     7681 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     7741 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     7801 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     7861 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     7921 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     7981 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     8041 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     8101 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     8161 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     8221 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     8281 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     8341 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     8401 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     8461 gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
