LOCUS       sequence-213191-        7397 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Pro-tRNA synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7397)
  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 7397)
  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: B6-2
FEATURES             Location/Qualifiers
     source          1..7397
                     /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             2080..2097
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2171..2189)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2185..2232
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2407..2412,2413..2429,2430..2435))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2462..2480)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2548..2652
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2653..2721,2722..3513)
                     /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(2871..2890)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3684..4272
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4173..4192
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4426..4443
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4458..4597
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4523..4542)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4683..4705
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4699..4890)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6202..7284)
                     /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     7246..7265
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7285..7362)
                     /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 atgcatcgta ctagccaata cctgctctcc actctcaagg agacacctgc cgacgccgag
      421 gtgatcagcc atcagctgat gctgcgcgcc gggatgatcc gcaagctggc ctccgggtta
      481 tatacctggc tgccgaccgg cgtgcgcgtt ctgaaaaaag tcgaaaacat cgtgcgtgaa
      541 gagatgaaca acgccggtgc gatcgaggtg tcgatgccgg tggttcagcc agccgatttg
      601 tggcaagaga gtggtcgttg ggaacagtac ggtccggaac tgctgcgttt tgttgaccgt
      661 ggcgagcgtc cgttcgtact cggcccaact catgaagaag ttatcactga cctgattcgt
      721 aacgagctta gctcttacaa acagctgccg ctgaacttct atcagatcca gaccaagttc
      781 cgcgacgaag tgcgtccgcg tttcggcgtc atgcgttccc gcgaattcct gatgaaagat
      841 gcttactctt tccatacttc tcaggaatcc ctgcaggaaa cctacgatgc aatgtatgcg
      901 gcctacagca aaatcttcag ccgcatgggg ctggatttcc gcgccgtaca agccgacacc
      961 ggttctatcg gcggcagcgc ctctcacgaa ttccaggtgc tggcgcagag cggtgaagac
     1021 gatgtggtct tctccgacac ctctgactat gcagcgaaca ttgaactggc agaagctatc
     1081 gcgccgaaag aaccgcgcgc tgctgctacc caggaaatga cgctggttga tacgccgaac
     1141 gcgaaaacca tcgcggaact ggttgaacag ttcaatctgc cgattgagaa aacggttaag
     1201 actctgctgg ttaaagcggt tgaaggcagc agcttcccgc aggttgcgct gctggtgcgc
     1261 ggtgatcacg agctgaacga agttaaagca gaaaaactgc cgcaggttgc aagcccgctg
     1321 actttcgcga ccgaagaaga aattcgtgcc gtggttaaag ccggtccggg ttcactgggt
     1381 ccggtaaaca tgccgattcc ggtggtgatt gaccgtaccg ttgcggcgat gagtgatttc
     1441 gctgctggtg ctaacatcga tggtaaacac tacttcggca tcaactggga tcgcgatgtc
     1501 gctaccccgg aagttgcaga tatccgtaac gtggtggctg gcgatccaag cccggatggc
     1561 cagggtaggc tgctgatcaa acgtggtatc gaagttggtc acatcttcca gctgggtacc
     1621 aagtactccg aagcactgaa agcctccgta cagggtgaag atggccgtaa ccaaatcctg
     1681 acgatgggtt gctacggtat cggggtaacg cgtgtggtag ctgcggcgat tgagcagaac
     1741 tacgacgaac gaggcatcgt atggcctgac gctatcgcgc cgttccaggt ggcgattctg
     1801 ccgatgaaca tgcacaaatc cttccgcgta caagagcttg ctgagaaact gtacagcgaa
     1861 ctgcgtgcac aaggtatcga agtgctgctg gatgaccgca aagagcgtcc gggcgtgatg
     1921 tttgctgata tggaactgat cggtattccg cacactattg tgctgggcga ccgtaacctc
     1981 gacaacgacg atatcgaata taaatatcgt cgcaacggcg agaaacagtt aattaagact
     2041 ggtgacatcg tcgaatatct ggtgaaacag attaaaggcc atcatcatca ccatcattaa
     2101 agcagaggcc atatgctcga ggatccggct gctaacaaag cccgaaagga agctgagttg
     2161 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2221 aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg cccggcagta
     2281 ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga
     2341 gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac
     2401 taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg aagacgaaag
     2461 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     2521 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2581 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2641 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2701 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2761 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2821 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     2881 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     2941 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3001 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3061 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3121 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     3181 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     3241 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3301 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3361 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3421 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3481 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3541 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3601 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3661 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3721 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3781 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3841 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3901 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3961 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4021 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4081 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4141 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4201 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4261 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4321 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4381 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4441 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4501 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     4561 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     4621 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4681 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     4741 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     4801 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     4861 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     4921 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     4981 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     5041 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     5101 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     5161 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     5221 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     5281 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     5341 gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct
     5401 ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct
     5461 ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg
     5521 gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag
     5581 acaaggtata gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg
     5641 gcataaatcg ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg
     5701 agcgatcctt gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc
     5761 aacgcgggca tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag
     5821 cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata
     5881 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     5941 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     6001 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     6061 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     6121 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     6181 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6241 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     6301 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     6361 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     6421 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     6481 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     6541 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     6601 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     6661 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     6721 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     6781 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     6841 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     6901 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     6961 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     7021 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     7081 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     7141 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     7201 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     7261 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     7321 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     7381 ccttatgcga ctcctgc
//
