LOCUS       sequence-389076-        4912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encodes two copies of the suppressor tRNA SupD that decodes 
            amber (UAG) codons as serine..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4912)
  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 4912)
  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: SupD v2
FEATURES             Location/Qualifiers
     source          1..4912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..17
                     /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     6..28
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(103..120)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      273..359
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      451..478
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             674..865
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(859..881)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    967..1106
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1022..1041
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1121..1138)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1292..1880)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1372..1391)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1991..2010)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2002..2817
                     /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(2075..2094)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        3484..3561
                     /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             3562..4644
                     /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(3581..3600)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4876..4881,4882..4898,4899..4905)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 ttgtgagcgg ataacaattt cacacaggaa acagaccatg cgcgccgtcg acctgcaggc
       61 atgcaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc
      121 agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc
      181 acctgacccc atgccgaact cagaagtgaa aggccgtagc gccgatggta gtgtggggtc
      241 tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag
      301 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
      361 cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc
      421 cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg
      481 cgtttctaca aactcttttt gtttgctagc ttttctctgg tcccgccgca tccataccgc
      541 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
      601 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
      661 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
      721 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
      781 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
      841 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
      901 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
      961 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     1021 gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat
     1081 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     1141 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     1201 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1261 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1321 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     1381 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1441 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     1501 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     1561 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     1621 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     1681 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     1741 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     1801 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     1861 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     1921 ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata
     1981 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg
     2041 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     2101 gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt
     2161 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     2221 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     2281 gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat
     2341 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     2401 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     2461 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     2521 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     2581 gtcgctcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     2641 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     2701 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     2761 gataatcctg atatgagtaa attgcagttt catttgatgc tcgatgagtt tttctaagaa
     2821 ttaattcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     2881 gcgcacattt ccccgaaaag tgccaccgcg gccgcgcttc tttgagcgaa cgatcaaaaa
     2941 taagtggcgc cccatcaaaa aaatattctc aacataaaaa actttgtgta atacttgtaa
     3001 cgctgaattc ggagagatgc cggagcggct gaacggaccg gtctctaaaa ccggagtagg
     3061 ggcaactcta ccgggggttc aaatccccct ctctccgcca ggatcctcta gagtcgacct
     3121 gcagatcctt agcgaaagct aaggattttt tttgccgcgg cgcttctttg agcgaacgat
     3181 caaaaataag tggcgcccca tcaaaaaaat attctcaaca taaaaaactt tgtgtaatac
     3241 ttgtaacgct gaattcggag agatgccgga gcggctgaac ggaccggtct ctaaaaccgg
     3301 agtaggggca actctaccgg gggttcaaat ccccctctct ccgccaggat cctctagagt
     3361 cgacctgcag atccttagcg aaagctaagg attttttttg cggccgcttt caccgtcatc
     3421 accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcatttac
     3481 gttgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     3541 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     3601 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     3661 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     3721 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     3781 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     3841 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     3901 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     3961 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatga ctctgaccag
     4021 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     4081 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     4141 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     4201 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     4261 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     4321 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     4381 gacgataccg aagacagctc atgttatatc ccgccgttag ccaccatcaa acaggatttt
     4441 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     4501 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     4561 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     4621 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc gcgaattgat
     4681 ctggtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc
     4741 atcggaagct gtggtatggc tgtgcaggtc gtagatcact gcataattcg tgtcgctcaa
     4801 ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata
     4861 ttctgaaatg agctgttgac aattaatcat ccggcccgta taatgtgtgg aa
//
