LOCUS       sequence-137236-        7038 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses truncated version of NLL-MetRS from endogenous MetG 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7038)
  AUTHORS   Mahdavi A, Szychowski J, Ngo JT, Sweredoski MJ, Graham RL, Hess S, 
            Schneewind O, Mazmanian SK, Tirrell DA
  TITLE     Identification of secreted bacterial proteins by noncanonical amino 
            acid tagging.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 Jan 7;111(1):433-8. doi: 
            10.1073/pnas.1301740111. Epub 2013 Dec 17.
  PUBMED    24347637
REFERENCE   2  (bases 1 to 7038)
  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: 
            "10.1073/pnas.1301740111"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2014-01-7- 7"; volume: "111"; issue: "1"; pages: "433-8"
COMMENT     Sequence Label: pAM1
FEATURES             Location/Qualifiers
     source          1..7038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(877..1692)
                     /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(877..1692)
                     /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     1600..1619
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1684..1703
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1764..1782
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1806..1820,1821..1826,1827..1843,1844..1850)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1826..1842
                     /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)."
     protein_bind    1858..1874
                     /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             1893..1904
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1923..1940
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2004..2098
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             4639..5298
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4705..4724)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      5363..5449
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5542..6624)
                     /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     6586..6605
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6625..6702)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(6786..6808)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6894..7034
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6949..6968
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatggcgtt aagggatttt ggtcatgaat taattcttag aaaaactcat cgagcatcaa
      901 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
      961 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     1021 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     1081 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     1141 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     1201 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     1261 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     1321 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     1381 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     1441 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     1501 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     1561 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     1621 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     1681 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     1741 tgaccatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcacctcg
     1801 agaaatcata aaaaatttat ttgctttgtg agcggataac aattataata gattcaattg
     1861 tgagcggata acaatttcac acagaattca ttaaagagga gaaattaact atgagaggat
     1921 cgcatcacca tcaccatcac ggatccgcat gcgagctcgg taccccgggt cgacctgcag
     1981 ccaagcttaa ttagctgagc ttggactcct gttgatagat ccagtaatga cctcagaact
     2041 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     2101 aagctagctc tagagacgtc cggccggagc tccaccgcgg tggcggccgc tctagagtca
     2161 cttacttaac attttcccat ttggtactat ctaacccctt ttcactatta agaagtaatg
     2221 cctactatga ctcaagtcgc gaagaaaatt ctggtgacgt gcgcaaaccc gtacgctaac
     2281 ggctcaatcc acctcggcca tatgctggag cacatccagg ctgatgtctg ggtccgttac
     2341 cagcgaatgc gcggccacga ggtcaacttc atctgcgccg acgatgccca cggtacaccg
     2401 atcatgctga aagctcagca gcttggtatc accccggagc agatgattgg cgaaatgagt
     2461 caggagcatc agactgattt cgcaggcttt aacatcagct atgacaacta tcactcgacg
     2521 cacagcgaag agaaccgcca gttgtcagaa cttatctact ctcgcctgaa agaaaacggt
     2581 tttattaaaa accgcaccat ctctcagctg tacgatccgg aaaaaggcat gttcctgccg
     2641 gaccgttttg tgaaaggcac ctgcccgaaa tgtaaatccc cggatcaata cggcgataac
     2701 tgcgaagtct gcggcgcgac ctacagcccg actgaactga tcgagccgaa atcggtggtt
     2761 tctggcgcta cgccggtaat gcgtgattct gaacacttct tctttgatct gccctctttc
     2821 agcgaaatgt tgcaggcatg gacccgcagc ggtgcgttgc aggagcaggt ggcaaataaa
     2881 atgcaggagt ggtttgaatc tggcctgcaa cagtgggata tctcccgcga cgccccttac
     2941 ttcggttttg aaattccgaa cgcgccgggc aaatatttct acgtctggct ggacgcaccg
     3001 attggcctga tgggttcttt caagaatctg tgcgacaagc gcggcgacag cgtaagcttc
     3061 gatgaatact ggaagaaaga ctccaccgcc gagctgtacc acttcatcgg taaagatatt
     3121 gtttacttcc tgagcctgtt ctggcctgcc atgctggaag gcagcaactt ccgcaagccg
     3181 tccaacctgt ttgttcatgg ctatgtgacg gtgaacggcg caaagatgtc caagtctcgc
     3241 ggcaccttta ttaaagccag cacctggctg aatcattttg acgcagacag cctgcgttac
     3301 tactacactg cgaaactctc ttcgcgcatt gatgatatcg atctcaacct ggaagatttc
     3361 gttcagcgtg tgaatgccga tatcgttaac aaagtggtta acctggcctc ccgtaatgcg
     3421 ggctttatca acaagcgttt tgacggcgtg ctggcaagcg aactggctga cccgcagttg
     3481 tacaaaacct tcactgatgc cgctgaagtg attggtgaag cgtgggaaag ccgtgaattt
     3541 ggtaaagccg tgcgcgaaat catggcgctg gctgatctgg ctaaccgcta tgtcgatgaa
     3601 caggctccgt gggtggtggc gaaacaggaa ggccgcgatg ccgacctgca ggcaatttgc
     3661 tcaatgggca tcaacctgtt ccgcgtgctg atgacttacc tgaagccggt actgccgaaa
     3721 ctgaccgagc gtgcagaagc attcctcaat acggaactga cctgggatgg tatccagcaa
     3781 ccgctgctgg gccacaaagt gaatccgttc aaggcgctgt ataaccgcat cgatatgagg
     3841 caggttgaag cactggtgga agcctctaaa tgagaagtaa aagccgctgc cgcgccggta
     3901 actggcccgc tggcagatga tccgattcag gaaaccatca cctttgacga cttcgctaaa
     3961 gttgacctgc gcgtggcgct gattgaaaac gcagagtttg ttgaaggttc tgacaaactg
     4021 ctgcgcctga cgctggatct cggcggtgaa aaacgcaatg tcttctccgg tattcgttct
     4081 gcttacccgg atccgcaggc actgattggt cgtcacacca ttatggtggc taacctggca
     4141 ccacgtaaaa tgcgcttcgg tatctctgaa ggcatggtga tggctgccgg tcctggcggg
     4201 aaagatattt tcctgctaag cccgatgccg gtgctaaacc gggtcatcag gtgaaataat
     4261 cccccttcaa ggcgctgcat cgacagcgcc ttttctttat aaattcctaa agttgttttc
     4321 ttgcgatttt gtctctctct aacccgcata aatactggta gcatctgcat tcaactggat
     4381 aaaattacag ggatgcagaa tgagacactt tatctatcag gacgaaaaat cacataaatt
     4441 cagggcagtt gagcaacagg gaaacgagtt gcatatcagt tggggaaaag ttggcaccaa
     4501 agggcaaagc cagataaaaa gtttttcaga tgctgcggca gcggcaaaag cggagcccac
     4561 ctcgaggggg ggcccggtac ccggccggac gtctctagag ctagcttggc gagattttca
     4621 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc
     4681 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac
     4741 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     4801 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaatttcgt
     4861 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     4921 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     4981 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     5041 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     5101 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc
     5161 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
     5221 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     5281 tggcagggcg gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggggta
     5341 atgactctct agcttgaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     5401 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccctctag
     5461 attacgtgca gtcgatgata agctgtcaaa catgagaatt gtgcctaatg agtgagctaa
     5521 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5581 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     5641 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5701 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     5761 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5821 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5881 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5941 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     6001 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     6061 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     6121 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     6181 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     6241 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     6301 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     6361 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     6421 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     6481 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     6541 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     6601 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     6661 tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg tcggaatttc gggcagcgtt
     6721 gggtcctggc cacgggtgcg catgatctag agctgcctcg cgcgtttcgg tgatgacggt
     6781 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     6841 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     6901 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     6961 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     7021 aataccgcat caggcgct
//
