LOCUS       sequence-430948-        5558 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of previously developed E. coli leucyl aminoacyl-tRNA 
            synthesis variant that is ""polyspecific"".
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5558)
  TITLE     Directed evolution of a bacterial leucyl tRNA in mammalian cells for
            enhanced noncanonical amino acid mutagenesis
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5558)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pIDTSmart-CMV-PLRS1
FEATURES             Location/Qualifiers
     source          1..5558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1..18)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1..17)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      58..645
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     546..565
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(657..684)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(708..1523)
                     /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="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1431..1450
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1524..1626)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(1627..1658)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     primer_bind     1706..1722
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1706..1722
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        1889..2268
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2347..2398
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     primer_bind     2349..2369
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2443..2462
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2443..2461
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2500..2509
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2506..5097
                     /codon_start=1
                     /product="aminoacyl-tRNA synthetase specific for the 
                     fluorescent amino acid Anap (Chatterjee et al., 2013)"
                     /label=AnapRS
                     /note="evolved from an E. coli leucyl-tRNA synthetase"
                     /translation="MEEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSILPYPS
                     GRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPAEGAAVKNNTAPAPWTYDN
                     IAYMKNQLKMLGFGYDWSRELATCTPEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQ
                     TVLANEQVIDGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDTVKTMQRN
                     WIGRSEGVEITFNVNDYDNTLTVYTTRPDAFMGCTYLAVAAGHPLAQKAAENNPELAAF
                     IDECRNTKVAEAEMATMEKKGVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPG
                     HDQRDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAI
                     ADKLTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDV
                     VMDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESSWIYARYTCPQYKEGMLDSEA
                     ANYWLPVDIAIGGIEHAIMGLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFYY
                     VGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQVMVE
                     RYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVAALNVDAL
                     TENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDGEQDRALMQEA
                     LLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRA
                     KITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVGGPV"
     primer_bind     complement(5135..5152)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5141..5365
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 actggccgtc gttttacacg ggtgggcctt tcttcggtag aaaatcaaag gatcttcttg
       61 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
      121 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaggtaac tggcttcagc
      181 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
      241 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
      301 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
      361 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
      421 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
      481 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
      541 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
      601 catcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgca
      661 gaaaggccca cccgaaggtg agccaggtga ttacatttag gtcctcatta gaaaaactca
      721 tcgagcatca agtgaaactg caatttattc atatcaggat tatcaatacc atatttttga
      781 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
      841 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
      901 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
      961 aatggcaaga gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     1021 tcatcaaaat cactcgcacc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     1081 cgaaatacgc gatcgccgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     1141 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     1201 tggaatgctg ttttccctgg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     1261 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag cctgaccatc
     1321 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     1381 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     1441 catttatacc catataaatc agcatccatg ttggaattta atcgcggctt caagcaagac
     1501 gtttcccgtt gaatatggct cattttagct tccttagctc ctgaaaatct cgataactca
     1561 aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc
     1621 cgatcaagtc aaaagcctcc ggtcggaggc ttttgacttt ctgctatgga ggtcaggtat
     1681 gatttaaatg gtcagtattg agcctcagga aacagctatg acatcaagct gactagataa
     1741 tctagctgat cgtggaccga tcatacgtat aatgccgtaa gatcacgggt cgcagcacag
     1801 ctcgcggtcc agtagtgatc gacactgctc gatccgctcg cacccctagg tgcgctgctt
     1861 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
     1921 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     1981 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     2041 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     2101 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     2161 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     2221 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     2281 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     2341 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
     2401 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
     2461 ggagacccaa gctggctagc gtttaaactt aagcttgccg ccaccatgga agagcaatac
     2521 cgcccggaag agatagaatc caaagtacag cttcattggg atgagaagcg cacatttgaa
     2581 gtaaccgaag acgagagcaa agagaagtat tactgcctgt ctatcctccc ctatccttct
     2641 ggtcgactac acatgggcca cgtacgtaac tacaccatcg gtgacgtgat cgcccgctac
     2701 cagcgtatgc tgggcaaaaa cgtcctgcag ccgatcggct gggacgcgtt tggtctgcct
     2761 gcggaaggcg cggcggtgaa aaacaacacc gctccggcac cgtggacgta cgacaacatc
     2821 gcgtatatga aaaaccagct caaaatgctg ggctttggtt atgactggag ccgcgagctg
     2881 gcaacctgta cgccggaata ctaccgttgg gaacagaaat tcttcaccga gctgtataaa
     2941 aaaggcctgg tatataagaa gacttctgcg gtcaactggt gtccgaacga ccagaccgta
     3001 ctggcgaacg aacaagttat cgacggctgc tgctggcgct gcgataccaa agttgaacgt
     3061 aaagagatcc cgcagtggtt tatcaaaatc actgcttacg ctgacgagct gctcaacgat
     3121 ctggataaac tggatcactg gccagacacc gttaaaacca tgcagcgtaa ctggatcggt
     3181 cgttccgaag gcgtggagat caccttcaac gttaacgact atgacaacac gctgaccgtt
     3241 tacactaccc gcccggacgc gtttatgggt tgtacctacc tggcggtagc tgcgggtcat
     3301 ccgctggcgc agaaagcggc ggaaaataat cctgaactgg cggcctttat tgacgaatgc
     3361 cgtaacacca aagttgccga agctgaaatg gcgacgatgg agaaaaaagg cgtcgatact
     3421 ggctttaaag cggttcaccc attaacgggc gaagaaattc ccgtttgggc agcaaacttc
     3481 gtattgatgg agtacggcac gggcgcagtt atggcggtac cggggcacga ccagcgcgac
     3541 tacgagtttg cctctaaata cggcctgaac atcaaaccgg ttatcctggc agctgacggc
     3601 tctgagccag atctttctca gcaagccctg actgaaaaag gcgtgctgtt caactctggc
     3661 gagttcaacg gtcttgacca tgaagcggcc ttcaacgcca tcgccgataa actgactgcg
     3721 atgggcgttg gcgagcgtaa agtgaactac cgcctgcgcg actggggtgt ttcccgtcag
     3781 cgttactggg gcgcgccgat tccgatggtg actctagaag acggtaccgt aatgccgacc
     3841 ccggacgacc agctgccggt gatcctgccg gaagatgtgg taatggacgg cattaccagc
     3901 ccgattaaag cagatccgga gtgggcgaaa actaccgtta acggtatgcc agcactgcgt
     3961 gaaaccgaca ctttcgacac ctttatggag tcctcctgga tctatgcgcg ctacacttgc
     4021 ccgcagtaca aagaaggtat gctggattcc gaagcggcta actactggct gccggtggat
     4081 atcgctattg gtggtattga acacgccatt atgggtctgc tctacttccg cttcttccac
     4141 aaactgatgc gtgatgcagg catggtgaac tctgacgaac cagcgaaaca gttgctgtgt
     4201 cagggtatgg tgctggcaga tgccttctac tatgttggcg aaaacggcga acgtaactgg
     4261 gtttccccgg ttgatgctat cgttgaacgt gacgagaaag gccgtatcgt gaaagcgaaa
     4321 gatgcggcag gccatgaact ggtttatacc ggcatgagca aaatgtccaa gtcgaagaac
     4381 aacggtatcg acccgcaggt gatggttgaa cgttacggcg cggacaccgt tcgtctgttt
     4441 atgatgtttg cttctccggc tgatatgact ctcgaatggc aggaatccgg tgtggaaggg
     4501 gctaaccgct tcctgaaacg tgtctggaaa ctggtttacg agcacacagc aaaaggtgat
     4561 gttgcggcac tgaacgttga tgcgctgact gaaaatcaga aagcgctgcg tcgcgatgtg
     4621 cataaaacga tcgctaaagt gaccgatgat atcggccgtc gtcagacctt caacaccgca
     4681 attgcggcga ttatggagct gatgaacaaa ctggcgaaag caccaaccga tggcgagcag
     4741 gatcgcgctc tgatgcagga agcactgctg gccgttgtcc gtatgcttaa cccgttcacc
     4801 ccgcacatct gcttcacgct gtggcaggaa ctgaaaggcg aaggcgatat cgacaacgcg
     4861 ccgtggccgg ttgctgacga aaaagcgatg gtggaagact ccacgctggt cgtggtgcag
     4921 gttaacggta aagtccgtgc caaaatcacc gttccggtgg acgcaacgga agaacaggtt
     4981 cgcgaacgtg ctggccagga acatctggta gcaaaatatc ttgatggcgt tactgtacgt
     5041 aaagtgattt acgtaccagg taaactcctc aatctggtcg ttggcgggcc cgtttaactc
     5101 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     5161 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     5221 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     5281 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     5341 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccctagc ggatcgacga
     5401 gagcagcgcg actggatctg tcgcccgtct caaacgcaac cctccggcgg tcgcatatca
     5461 ttcaggacga gcctcagact ccagcgtaac tggactgcaa tcaactcact ggctcacctt
     5521 ccggtccacg atcagctaga atcaagctga ctagataa
//
