LOCUS       sequence-442987-        7284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7284)
  AUTHORS   Park HS, Hohn MJ, Umehara T, Guo LT, Osborne EM, Benner J, Noren CJ,
            Rinehart J, Soll D
  TITLE     Expanding the genetic code of Escherichia coli with phosphoserine.
  JOURNAL   Science. 2011 Aug 26;333(6046):1151-4.
  PUBMED    21868676
REFERENCE   2  (bases 1 to 7284)
  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: "Science."; 
            date: "2011-08-26"; volume: "333(6046)"; pages: "1151-4"
COMMENT     Sequence Label: pKD-SepRS-EFSep
FEATURES             Location/Qualifiers
     source          1..7284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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..2301
                     /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(2316..2333)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2487..3075)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2567..2586)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3186..3205)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3197..4012
                     /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(3270..3289)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4187..4264
                     /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             4265..5347
                     /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(4284..4303)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5579..5584,5585..5601,5602..5608)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5616..5632
                     /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     5621..5643
                     /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 tcaacggggt 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 gtgaaaggcc 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 ttgcggtgtg aaataccgca cagatgcgta
     2281 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2341 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2401 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2461 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2521 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2581 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2641 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2701 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2761 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2821 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2881 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     2941 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3001 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3061 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3121 gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag
     3181 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt
     3241 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     3301 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa
     3361 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     3421 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     3481 gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga
     3541 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     3601 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     3661 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     3721 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcgc
     3781 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     3841 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     3901 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     3961 tcctgatatg agtaaattgc agtttcattt gatgctcgat gagtttttct aagaattaat
     4021 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4081 catttccccg aaaagtgcca ccgcggccgc tttcaccgtc atcaccgaaa cgcgcgaggc
     4141 agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca ccatcgaatg
     4201 gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     4261 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     4321 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     4381 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     4441 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     4501 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     4561 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     4621 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     4681 ctgcactaat gttccggcgt tatttcttga tgactctgac cagacaccca tcaacagtat
     4741 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     4801 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     4861 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     4921 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     4981 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     5041 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     5101 ctcatgttat atcccgccgt tagccaccat caaacaggat tttcgcctgc tggggcaaac
     5161 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     5221 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     5281 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     5341 gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt gatctggttt gacagcttat
     5401 catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat
     5461 ggctgtgcag gtcgtagatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg
     5521 gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt
     5581 gacaattaat catccggccc gtataatgtg tggaattgtg agcggataac aatttcacac
     5641 aggaaacaga ccatggcaat gtttaaaaga gaagaaatca ttgaaatggc caataaggac
     5701 tttgaaaaag catggatcga aactaaagac cttataaaag ctaaaaagat aaacgaaagt
     5761 tacccaagaa taaaaccagt ttttggaaaa acacaccctg taaatgacac tattgaaaat
     5821 ttaagacagg catatcttag aatgggtttt gaagaatata taaacccagt aattgtcgat
     5881 gaaagagata tttataaaca attcggccca gaagctatgg cagttttgga tagatgcttt
     5941 tatttagcgg gacttccaag acctgacgtt ggtttgagcg atgaaaaaat ttcacagatt
     6001 gaaaaacttg gaattaaagt ttctgagcac aaagaaagtt tacaaaaaat acttcacgga
     6061 tacaaaaaag gaactcttga tggtgacgat ttagttttag aaatttcaaa tgcacttgaa
     6121 atttcaagcg agatgggttt aaaaatttta gaagatgttt tcccagaatt taaggattta
     6181 accgcagttt cttcaaaatt aactttaaga agccacatga cttcaggatg gttccttact
     6241 gtttcagacc tcatgaacaa aaaacccttg ccatttaaac tcttttcaat cgatagatgt
     6301 tttagaagag aacaaaaaga agataaaagc cacttaatga cataccactc tgcatcctgt
     6361 gcaattgcag gtgaaggcgt ggatattaat gatggaaaag caattgcaga aggattatta
     6421 tcccaatttg gctttacaaa ctttaaattc attcctgatg aaaagaaaag taaatactac
     6481 acccctgaaa cacagactga agtttacgca taccacccaa aattaaaaga atggctcgaa
     6541 gttgctacat ttggagtata ttcgccagtt gcattaagca aatacggaat agatgtacct
     6601 gtaatgaatt tgggtcttgg tgttgaaaga cttgcaatga tttctggaaa tttcgcagat
     6661 gttcgagaaa tggtatatcc tcagttttac gaacacaaac ttaatgaccg gaatgtcgct
     6721 tcaatggtaa aactcgataa agttccagta atggatgaaa tttacgattt aacaaaagaa
     6781 ttaattgagt catgtgttaa aaacaaagat ttaaaatccc cttgtgaatt agctattgaa
     6841 aaaacgtttt catttggaaa aaccaagaaa aatgtaaaaa taaacatttt tgaaaaagaa
     6901 gaaggtaaaa atttactcgg accttcaatt ttaaacgaaa tctacgttta cgatggaaat
     6961 gtaattggaa ttcctgaaag ctttgacgga gtaaaagaag aatttaaaga cttcttagaa
     7021 aaaggaaaat cagaaggggt agcaacaggc attcgatata tcgatgcgct ttgctttaaa
     7081 attacttcaa aattagaaga agcatttgtg tcaaacacta ctgaattcaa agttaaagtt
     7141 ccaattgtca gaagtttaag cgacattaac ttaaaaatcg atgatatcgc attaaaacag
     7201 atcatgagca aaaataaagt aatcgacgtt agaggcccag tctttttaaa tgtcgaagta
     7261 aaaattgaat aagagctcgg tacc
//
