LOCUS       Exported                7584 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Phosphoserine orthogonal translation system.
ACCESSION   .
VERSION     .
KEYWORDS    SepOTS-gamma
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7584)
  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 7584)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7584
                     /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        4486..4563
                     /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             4564..5646
                     /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(4583..4602)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        5878..5907
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5915..5931
                     /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     5920..5942
                     /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 cgccctgttg cccgtctcac tggtgaaaag
     4141 aaaaaccacc ctggcgccgc ttctttgagc gaacgatcaa aaataagtgg cgccccatca
     4201 aaaaaatatt ctcaacataa aaaactttgt gtaatacttg taacgctgaa ttcgccgggg
     4261 tagtctaggg gttaggcagc ggactctaga tccgccttac gtgggttcaa atcccacccc
     4321 cggctccagg atcctctaga gtcgacctgc agatccttag cgaaagctaa ggattttttt
     4381 taagcttggc actggccgtc ggcggccgct ttcaccgtca tcaccgaaac gcgcgaggca
     4441 gcagatcaat tcgcgcgcga aggcgaagcg gcatgcattt acgttgacac catcgaatgg
     4501 tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     4561 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     4621 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     4681 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     4741 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     4801 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     4861 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     4921 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     4981 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     5041 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     5101 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     5161 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     5221 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     5281 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     5341 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     5401 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     5461 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     5521 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     5581 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     5641 cagtgagcgc aacgcaatta atgtgagtta gcgcgaattg atctggtttg acagcttatc
     5701 atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg
     5761 gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg
     5821 ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg
     5881 acaattaatc atccggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca
     5941 ggaaacagac ccatggcaat gtttaaaaga gaagaaatca ttgaaatggc caataaggac
     6001 tttgaaaaag catggatcga aactaaagac cttataaaag ctaaaaagat aaacgaaagt
     6061 tacccaagaa taaaaccagt ttttggaaaa acacaccctg taaatgacac tattgaaaat
     6121 ttaagacagg catatcttag aatgggtttt gaagaatata taaacccagt aattgtcgat
     6181 gaaagagata tttataaaca attcggccca gaagctatgg cagttttgga tagatgcttt
     6241 tatttagcgg gacttccaag acctgacgtt ggtttgagcg atgaaaaaat ttcacagatt
     6301 gaaaaacttg gaattaaagt ttctgagcac aaagaaagtt tacaaaaaat acttcacgga
     6361 tacaaaaaag gaactcttga tggtgacgat ttagttttag aaatttcaaa tgcacttgaa
     6421 atttcaagcg agatgggttt aaaaatttta gaagatgttt tcccagaatt taaggattta
     6481 accgcagttt cttcaaaatt aactttaaga agccacatga cttcaggatg gttccttact
     6541 gtttcagacc tcatgaacaa aaaacccttg ccatttaaac tcttttcaat cgatagatgt
     6601 tttagaagag aacaaaaaga agataaaagc cacttaatga cataccactc tgcatcctgt
     6661 gcaattgcag gtgaaggcgt ggatattaat gatggaaaag caattgcaga aggattatta
     6721 tcccaatttg gctttacaaa ctttaaattc attcctgatg aaaagaaaag taaatactac
     6781 acccctgaaa cacagactga agtttacgca taccacccaa aattaaaaga atggctcgaa
     6841 gttgctacat ttggagtata ttcgccagtt gcattaagca aatacggaat agatgtacct
     6901 gtaatgaatt tgggtcttgg tgttgaaaga cttgcaatga tttctggaaa tttcgcagat
     6961 gttcgagaaa tggtatatcc tcagttttac gaacacgaac ttaatgaccg ggatgtcgct
     7021 tcaatggtaa aactcgataa agttccagta atggatgaaa tttacgattt aacaaaagaa
     7081 ttaattgagt catgtgttaa aaacaaagat ttaaaatccc cttgtgaatt agctattgaa
     7141 aaaacgtttt catttggaaa aaccaagaaa aatgtaaaaa taaacatttt ttcaaaaata
     7201 gaaggtaaaa atttactcgg accttcaatt ttaaacgaaa tctacgttta cgatggaaat
     7261 gtaattggaa ttcctgaaag ctttgacgga gtaaaagaag aatttaaaga cttcttagaa
     7321 aaaggaaaat cagaaggggt agcaacaggc attcgatata tcgatgcgct ttgctttaaa
     7381 attacttcaa aattagaaga agcatttgtg tcaaacacta ctgaattcaa agttaaagtt
     7441 cgacgtgtca gaagtttaag cgacattaac ttaaaaatcg atgatatcgc aataaaacag
     7501 atcatgagca aaaataaagt aatcgacgtt agaggcccag tctttttaaa tgtcgaagta
     7561 aaaattgaat aagagctcgg tacc
//
