LOCUS       sequence-213177-        7082 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Asn-tRNA synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7082)
  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 7082)
  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: B4-3
FEATURES             Location/Qualifiers
     source          1..7082
                     /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"
     protein_bind    293..317
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     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(1856..1874)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1870..1917
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2092..2097,2098..2114,2115..2120))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2147..2165)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2233..2337
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2338..2406,2407..3198)
                     /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(2556..2575)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3369..3957
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3858..3877
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4111..4128
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4143..4282
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4208..4227)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4368..4390
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4384..4575)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5887..6969)
                     /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     6931..6950
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6970..7047)
                     /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 ggggaattgt
      301 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc
      361 atgcatcatc atcaccatca tttaagcgtt gtgcctgtag ccgacgtact ccagggccgt
      421 gtagccgttg acagcgaagt caccgtgcgc ggatgggtac gtacccgccg agattcaaaa
      481 gctggcatct ccttcctcgc cgtttatgac ggttcctgct ttgatcctgt acaggctgtc
      541 atcaataatt ctctgcccaa ttacaatgaa gacgtcctgc gtctgaccac cggctgctcg
      601 gtcattgtga cgggtaaagt cgtggcgtcg ccgggccagg ggcaacaatt tgaaattcag
      661 gccagcaagg ttgaagttgc tggttgggtt gaagatccag acacttaccc gatggcggca
      721 aaacgccaca gcattgagta tctgcgtgaa gtcgctcacc tgcgtccgcg cacaaacctg
      781 attggtgccg tcgcgcgcgt tcgccatacg ctggcgcagg cgctgcatcg cttctttaac
      841 gagcagggat tcttctgggt ttcaacgcca ctgattaccg catctgatac cgaaggtgca
      901 ggcgaaatgt tccgcgtttc tacgctggat ctggaaaacc tgccgcgtaa cgatcagggc
      961 aaagtggatt tcgacaaaga cttctttggt aaagagtctt tcctgaccgt atctggccag
     1021 ttgaacggcg aaacctacgc ttgcgcattg tccaaaattt ataccttcgg cccgactttc
     1081 cgtgctgaaa actccaacac cagccgtcac ctggcggaat tctggatgct ggagccggaa
     1141 gtggcgtttg ctaacctgaa cgatattgcg ggtctggctg aagccatgct gaaatatgtc
     1201 ttcaaagcgg ttctcgaaga acgcgctgac gacatgaaat tcttcgctga acgcgtagat
     1261 aaagatgccg tttcacgtct ggaacgcttc attgaagccg attttgcgca ggtggattat
     1321 accgacgcag tgaccattct cgaaaactgc ggcaggaagt ttgaaaaccc ggtttactgg
     1381 ggagtcgatc tctcttctga gcatgagcgt tatctggcgg aagaacactt taaagcaccg
     1441 gtagtggtta aaaactatcc gaaagatatt aaagcgttct atatgcgcct taacgaagac
     1501 ggtaaaaccg ttgcggctat ggacgttctg gctccgggca tcggtgagat cattggtggc
     1561 tcccagcgtg aagaacgtct ggacgtgctg gacgagcgta tgctggaaat gggcctgaat
     1621 aaagaagatt actggtggta tcgcgatctg cgtcgctacg gtactgttcc gcattcaggt
     1681 ttcggtcttg gttttgaacg tctgattgct tacgtaactg gcgtgcaaaa cgtacgtgat
     1741 gtgattccgt tcccacgtac tccgcgtaac gccagcttct attaaagcag aggccatatg
     1801 ctcgaggatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1861 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1921 aaaggaggaa ctatatccgg atatcccgca agaggcccgg cagtaccggc ataaccaagc
     1981 ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt
     2041 tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct
     2101 tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc
     2161 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     2221 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2281 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2341 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2401 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     2461 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     2521 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     2581 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     2641 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     2701 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     2761 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     2821 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     2881 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2941 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3001 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3061 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3121 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3181 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3241 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3301 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3361 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3421 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3481 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3541 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3601 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3661 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3721 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3781 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3841 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3901 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3961 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4021 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4081 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4141 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4201 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4261 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4321 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4381 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4441 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4501 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4561 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4621 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4681 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4741 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4801 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4861 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4921 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4981 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5041 tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc
     5101 gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt
     5161 ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc
     5221 gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
     5281 gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg
     5341 acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc
     5401 tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg
     5461 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac
     5521 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
     5581 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     5641 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     5701 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     5761 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     5821 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     5881 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5941 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     6001 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     6061 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     6121 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     6181 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6241 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     6301 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6361 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     6421 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     6481 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     6541 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     6601 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     6661 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6721 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6781 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     6841 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     6901 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     6961 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     7021 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     7081 gc
//
