LOCUS       sequence-213235-        6953 bp    DNA     circular SYN 14-APR-2025
DEFINITION  His-tRNA synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6953)
  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 6953)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: B8-3
FEATURES             Location/Qualifiers
     source          1..6953
                     /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             1636..1653
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1727..1745)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1741..1788
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1963..1968,1969..1985,1986..1991))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2018..2036)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2104..2208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2209..2277,2278..3069)
                     /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(2427..2446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3240..3828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3729..3748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3982..3999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4014..4153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4079..4098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4239..4261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4255..4446)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5758..6840)
                     /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     6802..6821
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6841..6918)
                     /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 atgcatgcaa aaaacattca agccattcgc ggcatgaacg attacctgcc tggcgaaacg
      421 gccatctggc agcgcattga aggcacactg aaaaacgtgc tcggcagcta cggttacagt
      481 gaaatccgct tgccgattgt agagcagacc ccgctattca aacgtgcgat tggtgaagtc
      541 accgacgtgg ttgaaaaaga gatgtacacc tttgaggatc gcaatggcga cagcctgact
      601 ctgcgccctg aagggacggc gggctgtgta cgcgccggca tcgagcatgg tcttctgtac
      661 aatcaggaac agcgtctgtg gtatatcggg ccgatgttcc gtcacgagcg tccgcagaaa
      721 gggcgttatc gtcagttcca tcagttgggc tgcgaagttt tcggtctgca aggtccggat
      781 atcgacgctg aactgattat gctcactgcc cgctggtggc gcgcgctggg tatttccgag
      841 cacgtaactc ttgagctgaa ctctatcggt tcgctggaag cacgcgccaa ttaccgcgat
      901 gcgctggtgg cattccttga gcagcataaa gaaaagctgg acgaagactg caaacgccgc
      961 atgtacacta acccgctgcg cgtgctggat tcaaaaaatc cggaagtgca ggcgcttctc
     1021 aacgacgctc cggcattagg tgactatctg gacgaggaat ctcgtgagca ttttgccggt
     1081 ctgtgcaaac tgctggagag cgcggggatc gcttacaccg taaaccagcg tctggtgcgt
     1141 ggtctggatt actacaaccg taccgttttc gagtgggtga ctaacagtct cggctcccag
     1201 ggcaccgtgt gtgcaggcgg tcgttatgac ggtcttgtgg aacaactggg cggtcgtgca
     1261 acaccggctg tcggttttgc tatgggcctc gaacgtcttg tattgttagt acaggccgtt
     1321 aatccggaat ttaaagccga tcctgttgtc gatatatacc tggtggcttc aggtgctgat
     1381 acacaatctg cggctatggc attagctgag cgtctgcgtg atgaattacc gggcgtgaaa
     1441 ttgatgacca accacggcgg cggcaacttt aagaaacagt ttgcccgtgc tgataaatgg
     1501 ggtgcccgcg ttgctgtggt gctgggtgag tctgaagtgg ctaacggcac agcagtagtg
     1561 aaggatttgc gctctggtga gcaaacggca gttgcgcagg atagcgtagc cgcgcatttg
     1621 cgcacgttac tgggtcatca tcatcaccat cattaaagca gaggccatat gctcgaggat
     1681 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1741 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1801 actatatccg gatatcccgc aagaggcccg gcagtaccgg cataaccaag cctatgccta
     1861 cagcatccag ggtgacggtg ccgaggatga cgatgagcgc attgttagat ttcatacacg
     1921 gtgcctgact gcgttagcaa tttaactgtg ataaactacc gcattaaagc ttatcgatga
     1981 taagctgtca aacatgagaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta
     2041 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     2101 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2161 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2221 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2281 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2341 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2401 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc
     2461 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2521 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2581 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     2641 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     2701 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg
     2761 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2821 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2881 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     2941 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3001 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3061 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3121 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3181 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3241 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3301 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3361 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3421 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3481 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3541 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3601 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3661 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3721 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3781 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3841 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3901 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3961 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4021 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4081 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4141 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4201 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4261 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4321 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     4381 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     4441 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     4501 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     4561 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     4621 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     4681 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     4741 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     4801 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     4861 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga
     4921 cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat
     4981 gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat
     5041 tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc
     5101 cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgcctaca
     5161 atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc
     5221 ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag ctgtccctga
     5281 tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg
     5341 gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag
     5401 acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     5461 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     5521 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     5581 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     5641 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     5701 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     5761 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5821 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     5881 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     5941 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     6001 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     6061 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     6121 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     6181 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     6241 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     6301 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     6361 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     6421 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     6481 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     6541 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     6601 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     6661 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     6721 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     6781 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     6841 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     6901 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgc
//
