LOCUS       sequence-326176-        8209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Phosphoserine orthogonal translation system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8209)
  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 8209)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2015-09-9"; volume: "6:8130. doi"; pages: " 
            10.1038/ncomms9130"
COMMENT     Sequence Label: SepOTS-kappa
FEATURES             Location/Qualifiers
     source          1..8209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..23,24..30)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    38..54
                     /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     43..65
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(3004..3021)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3174..3260
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3352..3379
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             3575..3766
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3760..3782)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3868..4007
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3923..3942
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4022..4039)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4193..4781)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4273..4292)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4892..4911)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4903..5718
                     /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
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVAHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMSKLQFHLMLDEFF"
     primer_bind     complement(4976..4995)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        6818..6895
                     /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             6896..7978
                     /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
                     YIPPLATIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(6915..6934)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatccggc ccgtataatg tgtggaattg tgagcggata acaatttcac
       61 acaggaaaca gaccatggca atgtttaaaa gagaagaaat cattgaaatg gccaataagg
      121 actttgaaaa agcatggatc gaaactaaag accttataaa agctaaaaag ataaacgaaa
      181 gttacccaag aataaaacca gtttttggaa aaacacaccc tgtaaatgac actattgaaa
      241 atttaagaca ggcatatctt agaatgggtt ttgaagaata tataaaccca gtaattgtcg
      301 atgaaagaga tatttataaa caattcggcc cagaagctat ggcagttttg gatagatgct
      361 tttatttagc gggacttcca agacctgacg ttggtttgag cgatgaaaaa atttcacaga
      421 ttgaaaaact tggaattaaa gtttctgagc acaaagaaag tttacaaaaa atacttcacg
      481 gatacaaaaa aggaactctt gatggtgacg atttagtttt agaaatttca aatgcacttg
      541 aaatttcaag cgagatgggt ttaaaaattt tagaagatgt tttcccagaa tttaaggatt
      601 taaccgcagt ttcttcaaaa ttaactttaa gaagccacat gacttcagga tggttcctta
      661 ctgtttcaga cctcatgaac aaaaaaccct tgccatttaa actcttttca atcgatagat
      721 gttttagaag agaacaaaaa gaagataaaa gccacttaat gacataccac tctgcatcct
      781 gtgcaattgc aggtgaaggc gtggatatta atgatggaaa agcaattgca gaaggattat
      841 tatcccaatt tggctttaca aactttaaat tcattcctga tgaaaagaaa agtaaatact
      901 acacccctga aacacagact gaagtttacg cataccaccc aaaattaaaa gaatggctcg
      961 aagttgctac atttggagta tattcgccag ttgcattaag caaatacgga atagatgtac
     1021 ctgtaatgaa tttgggtctt ggtgttgaaa gacttgcaat gatttctgga aatttcgcag
     1081 atgttcgaga aatggtatat cctcagtttt acgaacacaa acttaatgac cggaatgtcg
     1141 cttcaatggt aaaactcgat aaagttccag taatggatga aatttacgat ttaacaaaag
     1201 aattaattga gtcatgtgtt aaaaacaaag atttaaaatc cccttgtgaa ttagctattg
     1261 aaaaaacgtt ttcatttgga aaaaccaaga aaaatgtaaa aataaacatt tttgaaaaag
     1321 aagaaggtaa aaatttactc ggaccttcaa ttttaaacga aatctacgtt tacgatggaa
     1381 atgtaattgg aattcctgaa agctttgacg gagtaaaaga agaatttaaa gacttcttag
     1441 aaaaaggaaa atcagaaggg gtagcaacag gcattcgata tatcgatgcg ctttgcttta
     1501 aaattacttc aaaattagaa gaagcatttg tgtcaaacac tactgaattc aaagttaaag
     1561 ttccaattgt cagaagttta agcgacatta acttaaaaat cgatgatatc gcattaaaac
     1621 agatcatgag caaaaataaa gtaatcgacg ttagaggccc agtcttttta aatgtcgaag
     1681 taaaaattga ataagagctc ggtacctaaa gtatattagt taagtataag aaggagatat
     1741 acatatggga tccatgtcta aagaaaagtt tgaacgtaca aaaccgcacg ttaacgtcgg
     1801 tactatcggc cacgttgacc atggtaaaac aacgctgacc gctgcaatca ctaccgtact
     1861 ggctaaaacc tacggcggtg ctgctcgcgc attcgaccag atcgataacg cgccggaaga
     1921 aaaagctcgt ggtatcacca tcaacacttc tcgggttgaa tacgacaccc cgacccgtca
     1981 ctacgcacac gtagactgcc cggggcacgc cgactatgtt aaaaacatga tcaccggtgc
     2041 tgcgcagatg gacggcgcga tcctggtagt tgctgcgact gacggcccga tgccgcagac
     2101 tcgtgagcac atcctgctgg gtcgtcaggt aggcgttccg tacatcatcg tgttcctgaa
     2161 caaatgcgac atggttgatg acgaagagct gctggaactg gttgaaatgg aagttcgtga
     2221 acttctgtct cagtacgact tcccgggcga cgacactccg atcgttcgtg gttctgctct
     2281 gaaagcgctg gaaggcgacg cagagtggga agcgaaaatc ctggaactgg ctggcttcct
     2341 ggattcttac attccggaac cagagcgtgc gattgacaag ccgttcctgc tgccgatcaa
     2401 cggggtatac tccatctccg gtcgtggtac cgttgtttcg ggtcgtgtag aacgcggtat
     2461 catcaaagtt ggtgaagaag ttgaaatcgt tggtatcaaa gagactcaga agtctacctg
     2521 tactggcgtt gaaatgttcc gcaaactgct ggacgaaggc cgtgctggtg agtgggtagg
     2581 tgttctgctg cgtggtatca aacgtgaaga aatcgaacgt ggtcaggtac tggctaagcc
     2641 gggcaccatc aagccgcaca ccaagttcga atctgaagtg tacattctgt ccaaagatga
     2701 aggcggccgt catactccgt tcttcaaagg ctaccgtccg cagttctact tccgtactac
     2761 tgacgtgact ggtaccatcg aactgccgga aggcgtagag atggtaatgc cgggcgacaa
     2821 catcaaaatg gttgttaccc tgatccaccc gatcgcgatg gacgacggtc tgcgtttcgc
     2881 aatccgtgaa ggcggccgta ccgttggcgc gggcgttgta gcaaaagttc tgagctaatg
     2941 gcgcgccgtc gacctgcagg catgcaagct tggctgtttt ggcggatgag agaagatttt
     3001 cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg
     3061 cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga aaggccgtag
     3121 cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa
     3181 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     3241 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     3301 gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca
     3361 tcctgacgga tggccttttt gcgtttctac aaactctttt tgtttgctag cttttctctg
     3421 gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg
     3481 ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc
     3541 catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct
     3601 taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct
     3661 ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta
     3721 ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     3781 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     3841 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     3901 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattgc
     3961 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     4021 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4081 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4141 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4201 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4261 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4321 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4381 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4441 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4501 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4561 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4621 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4681 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4741 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4801 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca
     4861 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg
     4921 gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     4981 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
     5041 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     5101 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     5161 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca
     5221 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     5281 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     5341 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     5401 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     5461 ccattctcac cggattcagt cgtcgctcat ggtgatttct cacttgataa ccttattttt
     5521 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     5581 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     5641 ctttttcaaa aatatggtat tgataatcct gatatgagta aattgcagtt tcatttgatg
     5701 ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa
     5761 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaggcc gcgcttcttt
     5821 gagcgaacga tcaaaaataa gtggcgcccc atcaaaaaaa tattctcaac ataaaaaact
     5881 ttgtgtaata cttgtaacgc tgaattcgcc ggggtagtct aggggttagg cagcggactc
     5941 taaatccgcc ttacgtgggt tcaaatccca cccccggctc caggatcctc tagagtcgac
     6001 ctgcagatcc ttagcgaaag ctaaggattt tttttgccgc ggcgcttctt tgagcgaacg
     6061 atcaaaaata agtggcgccc catcaaaaaa atattctcaa cataaaaaac tttgtgtaat
     6121 acttgtaacg ctgaattcgc cggggtagtc taggggttag gcagcggact ctaaatccgc
     6181 cttacgtggg ttcaaatccc acccccggct ccaggatcct ctagagtcga cctgcagatc
     6241 cttagcgaaa gctaaggatt ttttttgcgg cccggccgcg cttctttgag cgaacgatca
     6301 aaaataagtg gcgccccatc aaaaaaatat tctcaacata aaaaactttg tgtaatactt
     6361 gtaacgctga attcgccggg gtagtctagg ggttaggcag cggactctaa atccgcctta
     6421 cgtgggttca aatcccaccc ccggctccag gatcctctag agtcgacctg cagatcctta
     6481 gcgaaagcta aggatttttt ttgccgcggc gcttctttga gcgaacgatc aaaaataagt
     6541 ggcgccccat caaaaaaata ttctcaacat aaaaaacttt gtgtaatact tgtaacgctg
     6601 aattcgccgg ggtagtctag gggttaggca gcggactcta aatccgcctt acgtgggttc
     6661 aaatcccacc cccggctcca ggatcctcta gagtcgacct gcagatcctt agcgaaagct
     6721 aaggattttt tttgcggccg ctttcaccgt catcaccgaa acgcgcgagg cagcagatca
     6781 attcgcgcgc gaaggcgaag cggcatgcat ttacgttgac accatcgaat ggtgcaaaac
     6841 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
     6901 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
     6961 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
     7021 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
     7081 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
     7141 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
     7201 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
     7261 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
     7321 tgttccggcg ttatttcttg atgactctga ccagacaccc atcaacagta ttattttctc
     7381 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
     7441 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
     7501 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
     7561 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
     7621 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     7681 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     7741 tatcccgccg ttagccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     7801 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     7861 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     7921 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     7981 gcaacgcaat taatgtaagt tagcgcgaat tgatctggtt tgacagctta tcatcgactg
     8041 cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca
     8101 ggtcgtagat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt
     8161 ttttgcgccg acatcataac ggttctggca aatattctga aatgagctg
//
