LOCUS       sequence-460364-        6415 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses mutant A. baumannii TrpRS from an inducible lac promoter 
            for proteome-wide incorporation of tryptophan analogues..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6415)
  TITLE     Proteome-wide incorporation of tryptophan analogues.
REFERENCE   2  (bases 1 to 6415)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pSGAb/pEvol-AbTrpRS(H14)
FEATURES             Location/Qualifiers
     source          1..6415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1920..1937
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     protein_bind    3035..3051
                     /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)."
     promoter        complement(join(3059..3065,3066..3081,3082..3087))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     promoter        3135..3212
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3213..4295
                     /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     complement(3232..3251)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      complement(4641..5229)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4721..4740)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5400..6215)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5400..6215)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6123..6142
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6216..6320)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6388..6406
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataggat tttaacattt tgcgttgttc caaaagttat caacagccta gaacgtcata
      241 ggaagcgatt acagacactt tatagctatc agcatgggaa cataagggca ggatgaaata
      301 tgggtcttaa aacgcaaatg gtgaggtttt agaggtattt tttgaagatg attaaggcgg
      361 tttgttttta aaatttttgg cggctctcag gctgcttaca ttttaaccag ttcagtgaaa
      421 agttcttttt cagcaaattt ctgtttagca ccatagctaa aacttgcgtg gaacatatta
      481 agaattgacc gaaatgacac aatctcaatt atattttttt tgaaaagttt tctttatcaa
      541 atattttaaa tcattgattt atatataagt atgcattcat tttaataatt aatctttatt
      601 taacaatgat ttatctatat tcaattgttt aattattctt actaatatta tctctatatc
      661 aatatttttt atttaaaaac atatgtttag tagtgctttt gattaaagta ccagagggag
      721 ggagcagagc tgaatgggaa atactcaccc tagagcgatt cttaaaaatc accctaaagt
      781 attcccattc gatgtaccgt cggtcggtcg ctttcgcatc agggatgaca tcactgtatc
      841 aagctgccac tgttatgatt acgattgata gcaccgcctg aacacgctca taaccgccaa
      901 ttaaatgact actcattgcg tccgctactc tgttcagttc ccctagtaat agcgtttttc
      961 cgatgtgtgc tagcgtcact gtacctcatc acccacacat ggacagattg agttacagac
     1021 attggctaaa tttttggggt ctatgcttga caaagcgagt taaaacctta gaatttaaga
     1081 caggtacatt aagcccctgt ggtgaaatca ttagcggtcg tcaaacctta atgctttcga
     1141 ttgccgatat gtcagtatcg aagatctgta ccccataaat tacggtaaag ccccaagcaa
     1201 ttgcaagggg cttttatctt ttttaacaaa aaaaatttat aaatcaggat tttataacac
     1261 taaataatcc aaagtacatg agtaagtcat gaccactcct gcgatgtgtg tagcactctg
     1321 agtatccgta ttatatcagt gatgtcatat acaaccacat aatgcggatg tatcactaac
     1381 tccctagtat ctttctgtct gcctgtgcgc cccatcagtc gattatcaac aagtaaatct
     1441 gtcttttctt caattaaatc atcaatttca atagctgcta tagggttgcg ttcttcaaga
     1501 taatcataga tatttctacg atcttggcta tgcggtgtga aataccgcac agatgcgtaa
     1561 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     1621 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     1681 gattaagttg ggtaacgcca gggtgattga tgaccgggag ctcacaaaca aggcccagtc
     1741 tttcgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcatggggag
     1801 accccacact accatcggcg ctacggcgtt tcacttctga gttcggcatg gggtcaggtg
     1861 ggaccaccgc gctactgccg ccaggcaaat tctgttttat cagaccgctt ctgcgttctg
     1921 atttaatctg tatcaggctg aaaatcttct ctcatccgcc aaaacagcca agctggagac
     1981 cgtttaaacg cggccgctta gaacttaaat aagcctaagc catctttaac ttcgtctaaa
     2041 gtctgctggg taatgattcg acatttatca gtaccttggc gtaaaacatc cataatgtag
     2101 tcaggatctt tagcaagttc ttctcggcgt tcacgtatag gtgtaattaa ttcttttagt
     2161 acaccttcaa gacgtttttt aactgtacca tcaccaagac caccacggcg gtagtgtgct
     2221 tttagctctt caacttcttc tttatttgga tcaaatgcat ctaaataagt aaatacaata
     2281 tttccttcaa cttgacccgg atcttcgatg cgaagatgat ttggatcggt atacatcgca
     2341 tttacagctt ttttgatgtc tttgtcagaa gcatttaaaa caatggtatt gcctaatgac
     2401 ttagacattt tcgctttacc atcaaaaccc ggtaaacgag ccatattaga aagcaatgct
     2461 ttacattcag gtaaaaggtc ttgaccaatt tgacggttta cacgacgcac gatttcgtta
     2521 gtttgctcaa tcattggaat ttgatcttcg ccgcatggtc ctacagtcgc tttaaaagcc
     2581 gtaatatcag ctgcttgtgc tacagggtag caaaggaaac cggcaggaat atcacgttca
     2641 aaaccacgca tttgaatttc agatttgatg gttgggttac gctccaaacg agctacagtt
     2701 acaaagttaa gatataacat tgtaagctca ttcagtgcag gcaagcatga ctgtacacaa
     2761 attgtggttt tagttggatc aatgcctacc gctaaatagt ctagagcaac ttctaaaatg
     2821 ttacggcgta ctttatctgg gttatcggca ttgtctgtta acgcttgtgc atctgctaat
     2881 aagagatgtt gatgatggct atcctgtaga cctacacgag aacgcaaaga accgacaaaa
     2941 tgcccgaggt gaagttgtcc ggttgggcgg tcgcctgcca aaataattgg acgctgatca
     3001 acattactca tgcggccgca cctcctttgt gaaattgtta tccgctcaca attccacaca
     3061 ttatacgagc cgatgattaa ttgtcaacag ctccctaatg caggagtcgc ataagggaga
     3121 gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc
     3181 gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg
     3241 cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg
     3301 tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca
     3361 accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca
     3421 gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac
     3481 tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg
     3541 cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg
     3601 accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg
     3661 tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg
     3721 gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa
     3781 gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa
     3841 ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca
     3901 tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg
     3961 cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gacatctcgg
     4021 tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca
     4081 aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg
     4141 gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc
     4201 tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     4261 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag
     4321 ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc
     4381 tccttccggt gggcgcgggg catgactaac atgagaatta caacttatat cgtatggggc
     4441 tgacttcagg tgctacattt gaagagataa attgcaagct tatcgatgaa gctgtcaaac
     4501 atgaggcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4561 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4621 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4681 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4741 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4801 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4861 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4921 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4981 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5041 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5101 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5161 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5221 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5281 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5341 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5401 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     5461 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     5521 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     5581 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     5641 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     5701 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     5761 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     5821 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     5881 tcttctaata cctggaatgc tgttttccca gggatcgcag tggtgagtaa ccatgcatca
     5941 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     6001 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     6061 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     6121 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     6181 ctagagcaag acgtttcccg ttgaatatgg ctcatactct tcctttttca atattattga
     6241 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6301 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     6361 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
