LOCUS Exported 7750 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Phosphoserine orthogonal translation system. ACCESSION . VERSION . KEYWORDS SepOTS-theta SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7750) AUTHORS Pirman NL, Barber KW, Aerni HR, Ma NJ, Haimovich AD, Rogulina S, Isaacs FJ, Rinehart J TITLE A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation. JOURNAL Nat Commun. 2015 Sep 9;6:8130. doi: 10.1038/ncomms9130. PUBMED 26350500 REFERENCE 2 (bases 1 to 7750) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7750 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(1298..1315) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 1468..1554 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 1646..1673 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" CDS 1869..2060 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind complement(2054..2076) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 2162..2304 /label=bom /note="basis of mobility region from pBR322" primer_bind 2217..2236 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(2319..2336) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2490..3078) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2570..2589) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind complement(3189..3208) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 3200..4015 /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 ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMSKLQFHLMLDEFF" primer_bind complement(3273..3292) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter 4652..4729 /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." CDS 4730..5812 /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 EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDDSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind complement(4749..4768) /label=LacI-R /note="LacI, reverse primer" promoter 6044..6073 /label=trc promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 6081..6097 /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)." primer_bind 6086..6108 /label=M13/pUC Reverse /note="In lacZ gene" ORIGIN 1 taaagtatat tagttaagta taagaaggag atatacatat gggatccatg tctaaagaaa 61 agtttgaacg tacaaaaccg cacgttaacg tcggtactat cggccacgtt gaccatggta 121 aaacaacgct gaccgctgca atcactaccg tactggctaa aacctacggc ggtgctgctc 181 gcgcattcga ccagatcgat aacgcgccgg aagaaaaagc tcgtggtatc accatcaaca 241 cttctcgggt tgaatacgac accccgaccc gtcactacgc acacgtagac tgcccggggc 301 acgccgacta tgttaaaaac atgatcaccg gtgctgcgca gatggacggc gcgatcctgg 361 tagttgctgc gactgacggc ccgatgccgc agactcgtga gcacatcctg ctgggtcgtc 421 aggtaggcgt tccgtacatc atcgtgttcc tgaacaaatg cgacatggtt gatgacgaag 481 agctgctgga actggttgaa atggaagttc gtgaacttct gtctcagtac gacttcccgg 541 gcgacgacac tccgatcgtt cgtggttctg ctctgaaagc gctggaaggc gacgcagagt 601 gggaagcgaa aatcctggaa ctggctggct tcctggattc ttacattccg gaaccagagc 661 gtgcgattga caagccgttc ctgctgccga tcaacggggt atactccatc tccggtcgtg 721 gtaccgttgt ttcgggtcgt gtagaacgcg gtatcatcaa agttggtgaa gaagttgaaa 781 tcgttggtat caaagagact cagaagtcta cctgtactgg cgttgaaatg ttccgcaaac 841 tgctggacga aggccgtgct ggtgagtggg taggtgttct gctgcgtggt atcaaacgtg 901 aagaaatcga acgtggtcag gtactggcta agccgggcac catcaagccg cacaccaagt 961 tcgaatctga agtgtacatt ctgtccaaag atgaaggcgg ccgtcatact ccgttcttca 1021 aaggctaccg tccgcagttc tacttccgta ctactgacgt gactggtacc atcgaactgc 1081 cggaaggcgt agagatggta atgccgggcg acaacatcaa aatggttgtt accctgatcc 1141 acccgatcgc gatggacgac ggtctgcgtt tcgcaatccg tgaaggcggc cgtaccgttg 1201 gcgcgggcgt tgtagcaaaa gttctgagct aatggcgcgc cgtcgacctg caggcatgca 1261 agcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac 1321 gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg 1381 accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc 1441 atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg 1501 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg 1561 ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca 1621 taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt 1681 ctacaaactc tttttgtttg ctagcttttc tctggtcccg ccgcatccat accgccagtt 1741 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga 1801 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg 1861 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc 1921 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct 1981 gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg 2041 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag 2101 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg 2161 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc 2221 atcagagcag attgtactga gagtgcacca tatatgcggt gtgaaatacc gcacagatgc 2281 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc 2341 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 2401 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 2461 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 2521 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 2581 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 2641 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 2701 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 2761 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 2821 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 2881 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 2941 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 3001 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 3061 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 3121 aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa 3181 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg 3241 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg 3301 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct 3361 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg 3421 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc 3481 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc 3541 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat 3601 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc 3661 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc 3721 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt 3781 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg 3841 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa 3901 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga 3961 taatcctgat atgagtaaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt 4021 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 4081 gcacatttcc ccgaaaagtg ccaccgcggc cgcgcttctt tgagcgaacg atcaaaaata 4141 agtggcgccc catcaaaaaa atattctcaa cataaaaaac tttgtgtaat acttgtaacg 4201 ctgaattcgc cggggtagtc taggggttag gcagcggact ctaaatccgc cttacgtggg 4261 ttcaaatccc acccccggct ccaggatcct ctagagtcga cctgcagatc cttagcgaaa 4321 gctaaggatt ttttttgccg cggcgcttct ttgagcgaac gatcaaaaat aagtggcgcc 4381 ccatcaaaaa aatattctca acataaaaaa ctttgtgtaa tacttgtaac gctgaattcg 4441 ccggggtagt ctaggggtta ggcagcggac tctaaatccg ccttacgtgg gttcaaatcc 4501 cacccccggc tccaggatcc tctagagtcg acctgcagat ccttagcgaa agctaaggat 4561 tttttttgcg gccgctttca ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc 4621 gcgcgaaggc gaagcggcat gcatttacgt tgacaccatc gaatggtgca aaacctttcg 4681 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt 4741 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt 4801 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga 4861 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat 4921 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa 4981 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt 5041 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat 5101 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc 5161 ggcgttattt cttgatgact ctgaccagac acccatcaac agtattattt tctcccatga 5221 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct 5281 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata 5341 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc 5401 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt 5461 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt 5521 tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc 5581 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt 5641 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt 5701 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 5761 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg 5821 caattaatgt gagttagcgc gaattgatct ggtttgacag cttatcatcg actgcacggt 5881 gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt 5941 aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc 6001 gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc 6061 ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagacccat 6121 ggcaatgttt aaaagagaag aaatcattga aatggccaat aaggactttg aaaaagcatg 6181 gatcgaaact aaagacctta taaaagctaa aaagataaac gaaagttacc caagaataaa 6241 accagttttt ggaaaaacac accctgtaaa tgacactatt gaaaatttaa gacaggcata 6301 tcttagaatg ggttttgaag aatatataaa cccagtaatt gtcgatgaaa gagatattta 6361 taaacaattc ggcccagaag ctatggcagt tttggataga tgcttttatt tagcgggact 6421 tccaagacct gacgttggtt tgagcgatga aaaaatttca cagattgaaa aacttggaat 6481 taaagtttct gagcacaaag aaagtttaca aaaaatactt cacggataca aaaaaggaac 6541 tcttgatggt gacgatttag ttttagaaat ttcaaatgca cttgaaattt caagcgagat 6601 gggtttaaaa attttagaag atgttttccc agaatttaag gatttaaccg cagtttcttc 6661 aaaattaact ttaagaagcc acatgacttc aggatggttc cttactgttt cagacctcat 6721 gaacaaaaaa cccttgccat ttaaactctt ttcaatcgat agatgtttta gaagagaaca 6781 aaaagaagat aaaagccact taatgacata ccactctgca tcctgtgcaa ttgcaggtga 6841 aggcgtggat attaatgatg gaaaagcaat tgcagaagga ttattatccc aatttggctt 6901 tacaaacttt aaattcattc ctgatgaaaa gaaaagtaaa tactacaccc ctgaaacaca 6961 gactgaagtt tacgcatacc acccaaaatt aaaagaatgg ctcgaagttg ctacatttgg 7021 agtatattcg ccagttgcat taagcaaata cggaatagat gtacctgtaa tgaatttggg 7081 tcttggtgtt gaaagacttg caatgatttc tggaaatttc gcagatgttc gagaaatggt 7141 atatcctcag ttttacgaac acaaacttaa tgaccggaat gtcgcttcaa tggtaaaact 7201 cgataaagtt ccagtaatgg atgaaattta cgatttaaca aaagaattaa ttgagtcatg 7261 tgttaaaaac aaagatttaa aatccccttg tgaattagct attgaaaaaa cgttttcatt 7321 tggaaaaacc aagaaaaatg taaaaataaa catttttgaa aaagaagaag gtaaaaattt 7381 actcggacct tcaattttaa acgaaatcta cgtttacgat ggaaatgtaa ttggaattcc 7441 tgaaagcttt gacggagtaa aagaagaatt taaagacttc ttagaaaaag gaaaatcaga 7501 aggggtagca acaggcattc gatatatcga tgcgctttgc tttaaaatta cttcaaaatt 7561 agaagaagca tttgtgtcaa acactactga attcaaagtt aaagttccaa ttgtcagaag 7621 tttaagcgac attaacttaa aaatcgatga tatcgcatta aaacagatca tgagcaaaaa 7681 taaagtaatc gacgttagag gcccagtctt tttaaatgtc gaagtaaaaa ttgaataaga 7741 gctcggtacc //