LOCUS       sequence-438919-        8439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used in phosphoprotein synthesis. Expresses the Sep-accepting tRNA 
            (tRNASep), the Sep-tRNA synthetase (SepRS) and an engineered EF-Tu 
            (EFSep).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8439)
  TITLE     Rinehart Lab Phosphoprotein synthesis components
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8439)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pKD‐SepRS-­EFSep-­5x tRNASep (B40 OTS)
FEATURES             Location/Qualifiers
     source          1..8439
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      complement(1403..1430)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1522..1608)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1761..1778
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     primer_bind     complement(4717..4739)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4728..4744
                     /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)."
     promoter        complement(join(4752..4758,4759..4775,4776..4781))
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(5013..6095)
                     /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     6057..6076
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6096..6173)
                     /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             complement(7503..8318)
                     /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     8226..8245
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8310..8329
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa gctagcaaac
     1381 aaaaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttaatttg
     1441 atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcgcaac
     1501 gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
     1561 gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctggcagt
     1621 tccctactct cgcatgggga gaccccacac taccatcggc gctacggcct ttcacttctg
     1681 agttcggcat ggggtcaggt gggaccaccg cgctactgcc gccaggcaaa ttctgtttta
     1741 tcagaccgct tctgcgttct gatttaatct gtatcaggct gaaaatcttc tctcatccgc
     1801 caaaacagcc aagcttgcat gcctgcaggt cgacggcgcg ccattagctc agaacttttg
     1861 ctacaacgcc cgcgccaacg gtacggccgc cttcacggat tgcgaaacgc agaccgtcgt
     1921 ccatcgcgat cgggtggatc agggtaacaa ccattttgat gttgtcgccc ggcattacca
     1981 tctctacgcc ttccggcagt tcgatggtac cagtcacgtc agtagtacgg aagtagaact
     2041 gcggacggta gcctttgaag aacggagtat gacggccgcc ttcatctttg gacagaatgt
     2101 acacttcaga ttcgaacttg gtgtgcggct tgatggtgcc cggcttagcc agtacctgac
     2161 cacgttcgat ttcttcacgt ttgataccac gcagcagaac acctacccac tcaccagcac
     2221 ggccttcgtc cagcagtttg cggaacattt caacgccagt acaggtagac ttctgagtct
     2281 ctttgatacc aacgatttca acttcttcac caactttgat gataccgcgt tctacacgac
     2341 ccgaaacaac ggtaccacga ccggagatgg agtatacccc gttgatcggc agcaggaacg
     2401 gcttgtcaat cgcacgctct ggttccggaa tgtaagaatc caggaagcca gccagttcca
     2461 ggattttcgc ttcccactct gcgtcgcctt ccagcgcttt cagagcagaa ccacgaacga
     2521 tcggagtgtc gtcgcccggg aagtcgtact gagacagaag ttcacgaact tccatttcaa
     2581 ccagttccag cagctcttcg tcatcaacca tgtcgcattt gttcaggaac acgatgatgt
     2641 acggaacgcc tacctgacga cccagcagga tgtgctcacg agtctgcggc atcgggccgt
     2701 cagtcgcagc aactaccagg atcgcgccgt ccatctgcgc agcaccggtg atcatgtttt
     2761 taacatagtc ggcgtgcccc gggcagtcta cgtgtgcgta gtgacgggtc ggggtgtcgt
     2821 attcaacccg agaagtgttg atggtgatac cacgagcttt ttcttccggc gcgttatcga
     2881 tctggtcgaa tgcgcgagca gcaccgccgt aggttttagc cagtacggta gtgattgcag
     2941 cggtcagcgt tgttttacca tggtcaacgt ggccgatagt accgacgtta acgtgcggtt
     3001 ttgtacgttc aaacttttct ttagacatgg atcccatatg tatatctcct tcttatactt
     3061 aactaatata ctttaggtac cgagctctta ttcaattttt acttcgacat ttaaaaagac
     3121 tgggcctcta acgtcgatta ctttattttt gctcatgatc tgttttaatg cgatatcatc
     3181 gatttttaag ttaatgtcgc ttaaacttct gacaattgga actttaactt tgaattcagt
     3241 agtgtttgac acaaatgctt cttctaattt tgaagtaatt ttaaagcaaa gcgcatcgat
     3301 atatcgaatg cctgttgcta ccccttctga ttttcctttt tctaagaagt ctttaaattc
     3361 ttcttttact ccgtcaaagc tttcaggaat tccaattaca tttccatcgt aaacgtagat
     3421 ttcgtttaaa attgaaggtc cgagtaaatt tttaccttct tctttttcaa aaatgtttat
     3481 ttttacattt ttcttggttt ttccaaatga aaacgttttt tcaatagcta attcacaagg
     3541 ggattttaaa tctttgtttt taacacatga ctcaattaat tcttttgtta aatcgtaaat
     3601 ttcatccatt actggaactt tatcgagttt taccattgaa gcgacattcc ggtcattaag
     3661 tttgtgttcg taaaactgag gatataccat ttctcgaaca tctgcgaaat ttccagaaat
     3721 cattgcaagt ctttcaacac caagacccaa attcattaca ggtacatcta ttccgtattt
     3781 gcttaatgca actggcgaat atactccaaa tgtagcaact tcgagccatt cttttaattt
     3841 tgggtggtat gcgtaaactt cagtctgtgt ttcaggggtg tagtatttac ttttcttttc
     3901 atcaggaatg aatttaaagt ttgtaaagcc aaattgggat aataatcctt ctgcaattgc
     3961 ttttccatca ttaatatcca cgccttcacc tgcaattgca caggatgcag agtggtatgt
     4021 cattaagtgg cttttatctt ctttttgttc tcttctaaaa catctatcga ttgaaaagag
     4081 tttaaatggc aagggttttt tgttcatgag gtctgaaaca gtaaggaacc atcctgaagt
     4141 catgtggctt cttaaagtta attttgaaga aactgcggtt aaatccttaa attctgggaa
     4201 aacatcttct aaaattttta aacccatctc gcttgaaatt tcaagtgcat ttgaaatttc
     4261 taaaactaaa tcgtcaccat caagagttcc ttttttgtat ccgtgaagta ttttttgtaa
     4321 actttctttg tgctcagaaa ctttaattcc aagtttttca atctgtgaaa ttttttcatc
     4381 gctcaaacca acgtcaggtc ttggaagtcc cgctaaataa aagcatctat ccaaaactgc
     4441 catagcttct gggccgaatt gtttataaat atctctttca tcgacaatta ctgggtttat
     4501 atattcttca aaacccattc taagatatgc ctgtcttaaa ttttcaatag tgtcatttac
     4561 agggtgtgtt tttccaaaaa ctggttttat tcttgggtaa ctttcgttta tctttttagc
     4621 ttttataagg tctttagttt cgatccatgc tttttcaaag tccttattgg ccatttcaat
     4681 gatttcttct cttttaaaca ttgccatggt ctgtttcctg tgtgaaattg ttatccgctc
     4741 acaattccac acattatacg ggccggatga ttaattgtca acagctcatt tcagaatatt
     4801 tgccagaacc gttatgatgt cggcgcaaaa aacattatcc agaacgggag tgcgccttga
     4861 gcgacacgaa ttatgcagtg atctacgacc tgcacagcca taccacagct tccgatggct
     4921 gcctgacgcc agaagcattg gtgcaccgtg cagtcgatga taagctgtca aaccagatca
     4981 attcgcgcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5041 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5101 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     5161 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     5221 tcctgtttga tggtggctaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     5281 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     5341 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     5401 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5461 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5521 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5581 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5641 tcagagtcat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5701 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5761 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5821 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5881 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5941 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     6001 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     6061 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     6121 tccgggcgct atcatgccat accgcgaaag gttttgcacc attcgatggt gtcaacgtaa
     6181 atgcatgccg cttcgccttc gcgcgcgaat tgatctgctg cctcgcgcgt ttcggtgatg
     6241 acggtgaaag cggccgcaaa aaaaatcctt agctttcgct aaggatctgc aggtcgactc
     6301 tagaggatcc tggagccggg ggtgggattt gaacccacgt aaggcggatc tagagtccgc
     6361 tgcctaaccc ctagactacc ccggcgaatt cagcgttaca agtattacac aaagtttttt
     6421 atgttgagaa tatttttttg atggggcgcc acttattttt gatcgttcgc tcaaagaagc
     6481 gcggccgcaa aaaaaatcct tagctttcgc taaggatctg caggtcgact ctagaggatc
     6541 ctggagccgg gggtgggatt tgaacccacg taaggcggat ctagagtccg ctgcctaacc
     6601 cctagactac cccggcgaat tcagcgttac aagtattaca caaagttttt tatgttgaga
     6661 atattttttt gatggggcgc cacttatttt tgatcgttcg ctcaaagaag cgcggccgca
     6721 aaaaaaatcc ttagctttcg ctaaggatct gcaggtcgac tctagaggat cctggagccg
     6781 ggggtgggat ttgaacccac gtaaggcgga tctagagtcc gctgcctaac ccctagacta
     6841 ccccggcgaa ttcagcgtta caagtattac acaaagtttt ttatgttgag aatatttttt
     6901 tgatggggcg ccacttattt ttgatcgttc gctcaaagaa gcgcggccgc aaaaaaaatc
     6961 cttagctttc gctaaggatc tgcaggtcga ctctagagga tcctggagcc gggggtggga
     7021 tttgaaccca cgtaaggcgg atctagagtc cgctgcctaa cccctagact accccggcga
     7081 attcagcgtt acaagtatta cacaaagttt tttatgttga gaatattttt ttgatggggc
     7141 gccacttatt tttgatcgtt cgctcaaaga agcgcggccg caaaaaaaat ccttagcttt
     7201 cgctaaggat ctgcaggtcg actctagagg atcctggagc cgggggtggg atttgaaccc
     7261 acgtaaggcg gatctagagt ccgctgccta acccctagac taccccggcg aattcagcgt
     7321 tacaagtatt acacaaagtt ttttatgttg agaatatttt tttgatgggg cgccacttat
     7381 ttttgatcgt tcgctcaaag aagcgcggcc gcggtggcac ttttcgggga aatgtgcgcg
     7441 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     7501 tcttagaaaa actcatcgag catcaaatga aactgcaatt tactcatatc aggattatca
     7561 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     7621 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     7681 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagcgacg
     7741 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     7801 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     7861 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     7921 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     7981 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     8041 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     8101 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     8161 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     8221 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     8281 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     8341 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     8401 ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
