LOCUS Exported 7897 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes Thr251Gly-EcPheRS Under IPTG-Inducible (PT5) Control. ACCESSION . VERSION . KEYWORDS pKPY514 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7897) AUTHORS Yuet KP, Doma MK, Ngo JT, Sweredoski MJ, Graham RL, Moradian A, Hess S, Schuman EM, Sternberg PW, Tirrell DA TITLE Cell-specific proteomic analysis in Caenorhabditis elegans. JOURNAL Proc Natl Acad Sci U S A. 2015 Feb 17. pii: 201421567. PUBMED 25691744 REFERENCE 2 (bases 1 to 7897) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7897 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 10..54 /label=T5 promoter /note="bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator" protein_bind 30..46 /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 62..78 /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 97..108 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 127..144 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" terminator 3564..3658 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS 3702..4361 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(3768..3787) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" terminator 4426..4512 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" CDS complement(4605..5687) /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 5649..5668 /label=LacI-R /note="LacI, reverse primer" promoter complement(5688..5765) /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(5849..5871) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 5957..6097 /label=bom /note="basis of mobility region from pBR322" primer_bind 6012..6031 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(6112..6129) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6283..6871) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6363..6382) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6978..7793) /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 7701..7720 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 7785..7804 /label=pENTR-R /note="pENTR vectors, reverse primer" primer_bind 7865..7883 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga 121 ggatcgcatc accatcacca tcacggatcc gcatgcatgt cacatctcgc agaactggtt 181 gccagtgcga aggcggccat tagccaggcg tcagatgttg ccgcgttaga taatgtgcgc 241 gtcgaatatt tgggtaaaaa agggcactta acccttcaga tgacgaccct gcgtgagctg 301 ccgccagaag agcgtccggc agctggtgcg gttatcaacg aagcgaaaga gcaggttcag 361 caggcgctga atgcgcgtaa agcggaactg gaaagcgctg cactgaatgc gcgtctggcg 421 gcggaaacga ttgatgtctc tctgccaggt cgtcgcattg aaaacggcgg tctgcatccg 481 gttacccgta ccatcgaccg tatcgaaagt ttcttcggtg agcttggctt taccgtggca 541 accgggccgg aaatcgaaga cgattatcat aacttcgatg ctctgaacat tcctggtcac 601 cacccggcgc gcgctgacca cgacactttc tggtttgaca ctacccgcct gctgcgtacc 661 cagacctctg gcgtacagat ccgcaccatg aaagcccagc agccaccgat tcgtatcatc 721 gcgcctggcc gtgtttatcg taacgactac gaccagactc acacgccgat gttccatcag 781 atggaaggtc tgattgttga taccaacatc agctttacca acctgaaagg cacgctgcac 841 gacttcctgc gtaacttctt tgaggaagat ttgcagattc gcttccgtcc ttcctacttc 901 ccgtttggag aaccttctgc agaagtggac gtcatgggta aaaacggtaa atggctggaa 961 gtgctgggct gcgggatggt gcatccgaac gtgttgcgta acgttggcat cgacccggaa 1021 gtttactctg gtttcgcctt cgggatgggg atggagcgtc tgactatgtt gcgttacggc 1081 gtcaccgacc tgcgttcatt cttcgaaaac gatctgcgtt tcctcaaaca gtttaaataa 1141 ggcaggaata gattatgaaa ttcagtgaac tgtggttacg cgaatgggtg aacccggcga 1201 ttgatagcga tgcgctggca aatcaaatca ctatggcggg cctggaagtt gacggtgtag 1261 aaccggttgc cggcagcttc cacggcgtgg tcgttggtga agtggttgag tgtgcgcagc 1321 atccgaacgc tgacaaactg cgtgtgacaa aagtgaatgt cggcggcgat cgcctgctgg 1381 acatcgtctg cggtgcgcca aactgccgtc agggcctgcg tgtagcggta gcgaccattg 1441 gtgctgttct gccgggtgat ttcaaaatta aagcggcgaa actgcgtggc gaaccgtctg 1501 aagggatgct gtgctccttc tctgaactgg gcatttctga cgatcacagc ggcattatcg 1561 aactgcctgc ggatgcgccg attggcaccg atatccgtga atacctgaaa cttgatgaca 1621 acaccatcga aatcagcgtg acgccaaacc gtgccgactg cttaggcatc attggtgttg 1681 cgcgtgacgt tgccgtgctg aaccagctgc cgctggttca accggaaatc gttccggttg 1741 gtgcgaccat cgacgacacg ctgccgatta cagtcgaagc gccggaagcc tgcccgcgtt 1801 atcttggccg tgtggtaaaa ggcattaacg ttaaagcgcc aactccgctg tggatgaaag 1861 aaaaactgcg tcgttgcggg atccgttcta tcgatgcagt tgttgacgtc accaactatg 1921 tgctgctcga actgggccag ccgatgcacg ctttcgataa agatcgcatt gaaggcggca 1981 ttgtggtgcg gatggcgaaa gagggcgaaa cgctggtgct gctcgacggt actgaagcga 2041 agctgaatgc tgacactctg gtcatcgccg accacaacaa ggcgctggcg atgggcggca 2101 tcttcggtgg cgaacactct ggcgtgaatg acgaaacaca aaacgtgctg ctggaatgcg 2161 cgttctttag cccgctgtct atcaccggtc gtgctcgtcg tcatggcctg cataccgatg 2221 cgtctcaccg ttatgagcgt ggcgttgatc cggcactgca gcacaaagcg atggaacgtg 2281 cgacccgtct gctgatcgac atctgcggtg gtgaggctgg cccggtaatt gatatcacca 2341 acgaagcaac gctgccgaag cgtgcaacca tcactctacg tcgtagcaaa ctggatcgcc 2401 tgatcggcca tcatattgcg gatgagcagg taactgacat tctgcgtcgt ctcggctgcg 2461 aagtgaccga aggcaaagac gaatggcagg cagttgcgcc gagctggcgt ttcgatatgg 2521 agattgaaga agatctggtt gaagaagtcg cgcgtgttta cggctacaac aacatcccgg 2581 atgagccggt acaggcaagc ctgattatgg gtactcaccg tgaagctgac ctgtcgctca 2641 agcgcgtgaa aacgctgctc aacgacaaag gctatcagga agtgatcacc tacagcttcg 2701 ttgatccgaa agtgcagcag atgatccatc caggcgttga agccttactg ctgccaagcc 2761 cgatctctgt tgaaatgtca gcaatgcgtc tttctctgtg gactggcctg ctggcaaccg 2821 tggtgtacaa ccagaaccgt cagcagaacc gtgtgcgcat tttcgaaagc ggtctgcgtt 2881 tcgtaccaga tactcaggca ccgttgggca ttcgtcagga tctgatgtta gccggtgtga 2941 tttgcggtaa ccgttacgaa gagcactgga acctggcaaa agagaccgtt gatttctatg 3001 atttgaaagg cgatcttgaa tccgttctcg acctgaccgg taaactgaat gaggttgagt 3061 tccgtgcaga agcgaatccg gcactgcatc cggggcaatc cgcagcgatt tatctgaaag 3121 gtgaacgtat tggttttgtt ggggttgttc atcctgaact ggaacgtaaa ctggatctta 3181 acggtcgcac tctggtgttc gaactggagt ggaacaagct cgcagaccgc gtggtgcctc 3241 aggcgcgcga gatttctcgc ttcccggcga accgtcgtga catcgcggtg gtggtcgcag 3301 aaaacgttcc cgcagcggat attttatccg aatgtaagaa agttggcgta aatcaggtag 3361 ttggcgtaaa cttatttgac gtgtaccgcg gtaagggtgt tgcggagggg tataagagcc 3421 tcgccataag cctgatcctg caagatacca gccgtacact cgaagaagag gagattgccg 3481 ctaccgtcgc caaatgtgta gaggcattaa aagagcgatt ccaggcatca ttgagggatt 3541 gaaagcttaa ttagctgagc ttggactcct gttgatagat ccagtaatga cctcagaact 3601 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc 3661 aagctagctt ggcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg 3721 gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt 3781 cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga 3841 ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga 3901 tgaatgctca tccggaattt cgtatggcaa tgaaagacgg tgagctggtg atatgggata 3961 gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga 4021 gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt 4081 acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag 4141 ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct 4201 tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc 4261 tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg 4321 aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa ggcagttatt 4381 ggtgccctta aacgcctggg gtaatgactc tctagcttga ggcatcaaat aaaacgaaag 4441 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg 4501 agtaggacaa atccgccctc tagattacgt gcagtcgatg ataagctgtc aaacatgaga 4561 attgtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc 4621 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 4681 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga 4741 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc 4801 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg 4861 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg 4921 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg 4981 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc 5041 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc 5101 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat 5161 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg 5221 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc 5281 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc 5341 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac 5401 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac 5461 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc 5521 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt 5581 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa 5641 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg 5701 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg 5761 gtgtcggaat ttcgggcagc gttgggtcct ggccacgggt gcgcatgatc tagagctgcc 5821 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 5881 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 5941 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg 6001 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat 6061 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac 6121 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 6181 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 6241 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 6301 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 6361 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 6421 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 6481 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 6541 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 6601 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 6661 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 6721 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 6781 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 6841 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 6901 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatggcgt taagggattt 6961 tggtcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc 7021 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac 7081 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt 7141 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa 7201 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag 7261 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg 7321 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa 7381 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt 7441 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg 7501 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata 7561 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct 7621 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc 7681 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg 7741 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc 7801 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccatga cattaaccta 7861 taaaaatagg cgtatcacga ggccctttcg tcttcac //