LOCUS       Exported                8443 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Phosphoserine orthogonal translation system.
ACCESSION   .
VERSION     .
KEYWORDS    SepOTS-nu
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8443)
  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 8443)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8443
                     /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        5345..5422
                     /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             5423..6505
                     /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(5442..5461)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        6737..6766
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6774..6790
                     /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     6779..6801
                     /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 ctagatccgc cttacgtggg
     4261 ttcaaatccc acccccggct ccaggatcct ctagagtcga cctgcagatc cttagcgaaa
     4321 gctaaggatt ttttttgcgg ccgcgcttct ttgagcgaac gatcaaaaat aagtggcgcc
     4381 ccatcaaaaa aatattctca acataaaaaa ctttgtgtaa tacttgtaac gctgaattcg
     4441 ccggggtagt ctaggggtta ggcagcggac tctagatccg ccttacgtgg gttcaaatcc
     4501 cacccccggc tccaggatcc tctagagtcg acctgcagat ccttagcgaa agctaaggat
     4561 tttttttgcg gccgcgcttc tttgagcgaa cgatcaaaaa taagtggcgc cccatcaaaa
     4621 aaatattctc aacataaaaa actttgtgta atacttgtaa cgctgaattc gccggggtag
     4681 tctaggggtt aggcagcgga ctctagatcc gccttacgtg ggttcaaatc ccacccccgg
     4741 ctccaggatc ctctagagtc gacctgcaga tccttagcga aagctaagga ttttttttgc
     4801 ggccgcgctt ctttgagcga acgatcaaaa ataagtggcg ccccatcaaa aaaatattct
     4861 caacataaaa aactttgtgt aatacttgta acgctgaatt cgccggggta gtctaggggt
     4921 taggcagcgg actctagatc cgccttacgt gggttcaaat cccacccccg gctccaggat
     4981 cctctagagt cgacctgcag atccttagcg aaagctaagg attttttttg cggccgcgct
     5041 tctttgagcg aacgatcaaa aataagtggc gccccatcaa aaaaatattc tcaacataaa
     5101 aaactttgtg taatacttgt aacgctgaat tcgccggggt agtctagggg ttaggcagcg
     5161 gactctagat ccgccttacg tgggttcaaa tcccaccccc ggctccagga tcctctagag
     5221 tcgacctgca gatccttagc gaaagctaag gatttttttt gcggccgctt tcaccgtcat
     5281 caccgaaacg cgcgaggcag cagatcaatt cgcgcgcgaa ggcgaagcgg catgcattta
     5341 cgttgacacc atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     5401 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     5461 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     5521 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     5581 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     5641 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     5701 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     5761 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     5821 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     5881 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     5941 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     6001 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     6061 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     6121 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     6181 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     6241 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     6301 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     6361 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     6421 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     6481 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag cgcgaattga
     6541 tctggtttga cagcttatca tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc
     6601 catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca
     6661 aggcgcactc ccgttctgga taatgttttt tgcgccgaca tcataacggt tctggcaaat
     6721 attctgaaat gagctgttga caattaatca tccggctcgt ataatgtgtg gaattgtgag
     6781 cggataacaa tttcacacag gaaacagacc catggcaatg tttaaaagag aagaaatcat
     6841 tgaaatggcc aataaggact ttgaaaaagc atggatcgaa actaaagacc ttataaaagc
     6901 taaaaagata aacgaaagtt acccaagaat aaaaccagtt tttggaaaaa cacaccctgt
     6961 aaatgacact attgaaaatt taagacaggc atatcttaga atgggttttg aagaatatat
     7021 aaacccagta attgtcgatg aaagagatat ttataaacaa ttcggcccag aagctatggc
     7081 agttttggat agatgctttt atttagcggg acttccaaga cctgacgttg gtttgagcga
     7141 tgaaaaaatt tcacagattg aaaaacttgg aattaaagtt tctgagcaca aagaaagttt
     7201 acaaaaaata cttcacggat acaaaaaagg aactcttgat ggtgacgatt tagttttaga
     7261 aatttcaaat gcacttgaaa tttcaagcga gatgggttta aaaattttag aagatgtttt
     7321 cccagaattt aaggatttaa ccgcagtttc ttcaaaatta actttaagaa gccacatgac
     7381 ttcaggatgg ttccttactg tttcagacct catgaacaaa aaacccttgc catttaaact
     7441 cttttcaatc gatagatgtt ttagaagaga acaaaaagaa gataaaagcc acttaatgac
     7501 ataccactct gcatcctgtg caattgcagg tgaaggcgtg gatattaatg atggaaaagc
     7561 aattgcagaa ggattattat cccaatttgg ctttacaaac tttaaattca ttcctgatga
     7621 aaagaaaagt aaatactaca cccctgaaac acagactgaa gtttacgcat accacccaaa
     7681 attaaaagaa tggctcgaag ttgctacatt tggagtatat tcgccagttg cattaagcaa
     7741 atacggaata gatgtacctg taatgaattt gggtcttggt gttgaaagac ttgcaatgat
     7801 ttctggaaat ttcgcagatg ttcgagaaat ggtatatcct cagttttacg aacacgaact
     7861 taatgaccgg gatgtcgctt caatggtaaa actcgataaa gttccagtaa tggatgaaat
     7921 ttacgattta acaaaagaat taattgagtc atgtgttaaa aacaaagatt taaaatcccc
     7981 ttgtgaatta gctattgaaa aaacgttttc atttggaaaa accaagaaaa atgtaaaaat
     8041 aaacattttt tcaaaaatag aaggtaaaaa tttactcgga ccttcaattt taaacgaaat
     8101 ctacgtttac gatggaaatg taattggaat tcctgaaagc tttgacggag taaaagaaga
     8161 atttaaagac ttcttagaaa aaggaaaatc agaaggggta gcaacaggca ttcgatatat
     8221 cgatgcgctt tgctttaaaa ttacttcaaa attagaagaa gcatttgtgt caaacactac
     8281 tgaattcaaa gttaaagttc gacgtgtcag aagtttaagc gacattaact taaaaatcga
     8341 tgatatcgca ataaaacaga tcatgagcaa aaataaagta atcgacgtta gaggcccagt
     8401 ctttttaaat gtcgaagtaa aaattgaata agagctcggt acc
//
