LOCUS       Exported                7750 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Phosphoserine orthogonal translation system.
ACCESSION   .
VERSION     .
KEYWORDS    SepOTS-eta
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 13, 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
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     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 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 cgcaaaaaaa atccttagct ttcgctaagg
     4141 atctgcaggt cgactctaga ggatcctgga gccgggggtg ggatttgaac ccacgtaagg
     4201 cggatctaga gtccgctgcc taacccctag actaccccgg cgaattcagc gttacaagta
     4261 ttacacaaag ttttttatgt tgagaatatt tttttgatgg ggcgccactt atttttgatc
     4321 gttcgctcaa agaagcgcgg ccgcaaaaaa aatccttagc tttcgctaag gatctgcagg
     4381 tcgactctag aggatcctgg agccgggggt gggatttgaa cccacgtaag gcggatctag
     4441 agtccgctgc ctaaccccta gactaccccg gcgaattcag cgttacaagt attacacaaa
     4501 gttttttatg ttgagaatat ttttttgatg gggcgccact tatttttgat cgttcgctca
     4561 aagaagcgcg 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 cttgatgtct 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 acgaacttaa tgaccgggat gtcgcttcaa tggtaaaact
     7201 cgataaagtt ccagtaatgg atgaaattta cgatttaaca aaagaattaa ttgagtcatg
     7261 tgttaaaaac aaagatttaa aatccccttg tgaattagct attgaaaaaa cgttttcatt
     7321 tggaaaaacc aagaaaaatg taaaaataaa cattttttca aaaatagaag 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 aaagttcgac gtgtcagaag
     7621 tttaagcgac attaacttaa aaatcgatga tatcgcaata aaacagatca tgagcaaaaa
     7681 taaagtaatc gacgttagag gcccagtctt tttaaatgtc gaagtaaaaa ttgaataaga
     7741 gctcggtacc
//
