LOCUS       Exported                7519 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Phosphoserine orthogonal translation system.
ACCESSION   .
VERSION     .
KEYWORDS    SepOTS-epsilon
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7519)
  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 7519)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7519
                     /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        4421..4498
                     /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             4499..5581
                     /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(4518..4537)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        5813..5842
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5850..5866
                     /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     5855..5877
                     /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 tcgtcggggt 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 ttttttgcgg ccgctttcac cgtcatcacc gaaacgcgcg aggcagcaga
     4381 tcaattcgcg cgcgaaggcg aagcggcatg catttacgtt gacaccatcg aatggtgcaa
     4441 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     4501 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     4561 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     4621 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     4681 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     4741 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     4801 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     4861 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     4921 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     4981 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     5041 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     5101 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     5161 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     5221 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     5281 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     5341 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     5401 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     5461 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     5521 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     5581 agcgcaacgc aattaatgtg agttagcgcg aattgatctg gtttgacagc ttatcatcga
     5641 ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt
     5701 gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat
     5761 gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat
     5821 taatcatccg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     5881 cagacccatg gcaatgttta aaagagaaga aatcattgaa atggccaata aggactttga
     5941 aaaagcatgg atcgaaacta aagaccttat aaaagctaaa aagataaacg aaagttaccc
     6001 aagaataaaa ccagtttttg gaaaaacaca ccctgtaaat gacactattg aaaatttaag
     6061 acaggcatat cttagaatgg gttttgaaga atatataaac ccagtaattg tcgatgaaag
     6121 agatatttat aaacaattcg gcccagaagc tatggcagtt ttggatagat gcttttattt
     6181 agcgggactt ccaagacctg acgttggttt gagcgatgaa aaaatttcac agattgaaaa
     6241 acttggaatt aaagtttctg agcacaaaga aagtttacaa aaaatacttc acggatacaa
     6301 aaaaggaact cttgatggtg acgatttagt tttagaaatt tcaaatgcac ttgaaatttc
     6361 aagcgagatg ggtttaaaaa ttttagaaga tgttttccca gaatttaagg atttaaccgc
     6421 agtttcttca aaattaactt taagaagcca catgacttca ggatggttcc ttactgtttc
     6481 agacctcatg aacaaaaaac ccttgccatt taaactcttt tcaatcgata gatgttttag
     6541 aagagaacaa aaagaagata aaagccactt aatgacatac cactctgcat cctgtgcaat
     6601 tgcaggtgaa ggcgtggata ttaatgatgg aaaagcaatt gcagaaggat tattatccca
     6661 atttggcttt acaaacttta aattcattcc tgatgaaaag aaaagtaaat actacacccc
     6721 tgaaacacag actgaagttt acgcatacca cccaaaatta aaagaatggc tcgaagttgc
     6781 tacatttgga gtatattcgc cagttgcatt aagcaaatac ggaatagatg tacctgtaat
     6841 gaatttgggt cttggtgttg aaagacttgc aatgatttct ggaaatttcg cagatgttcg
     6901 agaaatggta tatcctcagt tttacgaaca cgaacttaat gaccgggatg tcgcttcaat
     6961 ggtaaaactc gataaagttc cagtaatgga tgaaatttac gatttaacaa aagaattaat
     7021 tgagtcatgt gttaaaaaca aagatttaaa atccccttgt gaattagcta ttgaaaaaac
     7081 gttttcattt ggaaaaacca agaaaaatgt aaaaataaac attttttcaa aaatagaagg
     7141 taaaaattta ctcggacctt caattttaaa cgaaatctac gtttacgatg gaaatgtaat
     7201 tggaattcct gaaagctttg acggagtaaa agaagaattt aaagacttct tagaaaaagg
     7261 aaaatcagaa ggggtagcaa caggcattcg atatatcgat gcgctttgct ttaaaattac
     7321 ttcaaaatta gaagaagcat ttgtgtcaaa cactactgaa ttcaaagtta aagttcgacg
     7381 tgtcagaagt ttaagcgaca ttaacttaaa aatcgatgat atcgcaataa aacagatcat
     7441 gagcaaaaat aaagtaatcg acgttagagg cccagtcttt ttaaatgtcg aagtaaaaat
     7501 tgaataagag ctcggtacc
//
