LOCUS       sequence-102909-        7897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Thr251Gly-EcPheRS Under IPTG-Inducible (PT5) Control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7897)
  AUTHORS   Yuet KP, Doma MK, Ngo JT, Sweredoski MJ, Graham RL, Moradian A, Hess
            S, Schuman EM, Sternberg PW, Tirrell DA
  TITLE     Cell-specific proteomic analysis in Caenorhabditis elegans.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Feb 17. pii: 201421567.
  PUBMED    25691744
REFERENCE   2  (bases 1 to 7897)
  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: "Proc Natl 
            Acad Sci U S A. 2015 Feb 17. pii: 201421567."
COMMENT     Sequence Label: pKPY514
FEATURES             Location/Qualifiers
     source          1..7897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3564..3658
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             3702..4361
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3768..3787)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      4426..4512
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4605..5687)
                     /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     5649..5668
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5688..5765)
                     /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."
     primer_bind     complement(5849..5871)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5957..6097
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6012..6031
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6112..6129)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6283..6871)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6363..6382)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6978..7793)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6978..7793)
                     /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
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     7701..7720
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7785..7804
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     7865..7883
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatcgcatc accatcacca tcacggatcc gcatgcatgt cacatctcgc agaactggtt
      181 gccagtgcga aggcggccat tagccaggcg tcagatgttg ccgcgttaga taatgtgcgc
      241 gtcgaatatt tgggtaaaaa agggcactta acccttcaga tgacgaccct gcgtgagctg
      301 ccgccagaag agcgtccggc agctggtgcg gttatcaacg aagcgaaaga gcaggttcag
      361 caggcgctga atgcgcgtaa agcggaactg gaaagcgctg cactgaatgc gcgtctggcg
      421 gcggaaacga ttgatgtctc tctgccaggt cgtcgcattg aaaacggcgg tctgcatccg
      481 gttacccgta ccatcgaccg tatcgaaagt ttcttcggtg agcttggctt taccgtggca
      541 accgggccgg aaatcgaaga cgattatcat aacttcgatg ctctgaacat tcctggtcac
      601 cacccggcgc gcgctgacca cgacactttc tggtttgaca ctacccgcct gctgcgtacc
      661 cagacctctg gcgtacagat ccgcaccatg aaagcccagc agccaccgat tcgtatcatc
      721 gcgcctggcc gtgtttatcg taacgactac gaccagactc acacgccgat gttccatcag
      781 atggaaggtc tgattgttga taccaacatc agctttacca acctgaaagg cacgctgcac
      841 gacttcctgc gtaacttctt tgaggaagat ttgcagattc gcttccgtcc ttcctacttc
      901 ccgtttggag aaccttctgc agaagtggac gtcatgggta aaaacggtaa atggctggaa
      961 gtgctgggct gcgggatggt gcatccgaac gtgttgcgta acgttggcat cgacccggaa
     1021 gtttactctg gtttcgcctt cgggatgggg atggagcgtc tgactatgtt gcgttacggc
     1081 gtcaccgacc tgcgttcatt cttcgaaaac gatctgcgtt tcctcaaaca gtttaaataa
     1141 ggcaggaata gattatgaaa ttcagtgaac tgtggttacg cgaatgggtg aacccggcga
     1201 ttgatagcga tgcgctggca aatcaaatca ctatggcggg cctggaagtt gacggtgtag
     1261 aaccggttgc cggcagcttc cacggcgtgg tcgttggtga agtggttgag tgtgcgcagc
     1321 atccgaacgc tgacaaactg cgtgtgacaa aagtgaatgt cggcggcgat cgcctgctgg
     1381 acatcgtctg cggtgcgcca aactgccgtc agggcctgcg tgtagcggta gcgaccattg
     1441 gtgctgttct gccgggtgat ttcaaaatta aagcggcgaa actgcgtggc gaaccgtctg
     1501 aagggatgct gtgctccttc tctgaactgg gcatttctga cgatcacagc ggcattatcg
     1561 aactgcctgc ggatgcgccg attggcaccg atatccgtga atacctgaaa cttgatgaca
     1621 acaccatcga aatcagcgtg acgccaaacc gtgccgactg cttaggcatc attggtgttg
     1681 cgcgtgacgt tgccgtgctg aaccagctgc cgctggttca accggaaatc gttccggttg
     1741 gtgcgaccat cgacgacacg ctgccgatta cagtcgaagc gccggaagcc tgcccgcgtt
     1801 atcttggccg tgtggtaaaa ggcattaacg ttaaagcgcc aactccgctg tggatgaaag
     1861 aaaaactgcg tcgttgcggg atccgttcta tcgatgcagt tgttgacgtc accaactatg
     1921 tgctgctcga actgggccag ccgatgcacg ctttcgataa agatcgcatt gaaggcggca
     1981 ttgtggtgcg gatggcgaaa gagggcgaaa cgctggtgct gctcgacggt actgaagcga
     2041 agctgaatgc tgacactctg gtcatcgccg accacaacaa ggcgctggcg atgggcggca
     2101 tcttcggtgg cgaacactct ggcgtgaatg acgaaacaca aaacgtgctg ctggaatgcg
     2161 cgttctttag cccgctgtct atcaccggtc gtgctcgtcg tcatggcctg cataccgatg
     2221 cgtctcaccg ttatgagcgt ggcgttgatc cggcactgca gcacaaagcg atggaacgtg
     2281 cgacccgtct gctgatcgac atctgcggtg gtgaggctgg cccggtaatt gatatcacca
     2341 acgaagcaac gctgccgaag cgtgcaacca tcactctacg tcgtagcaaa ctggatcgcc
     2401 tgatcggcca tcatattgcg gatgagcagg taactgacat tctgcgtcgt ctcggctgcg
     2461 aagtgaccga aggcaaagac gaatggcagg cagttgcgcc gagctggcgt ttcgatatgg
     2521 agattgaaga agatctggtt gaagaagtcg cgcgtgttta cggctacaac aacatcccgg
     2581 atgagccggt acaggcaagc ctgattatgg gtactcaccg tgaagctgac ctgtcgctca
     2641 agcgcgtgaa aacgctgctc aacgacaaag gctatcagga agtgatcacc tacagcttcg
     2701 ttgatccgaa agtgcagcag atgatccatc caggcgttga agccttactg ctgccaagcc
     2761 cgatctctgt tgaaatgtca gcaatgcgtc tttctctgtg gactggcctg ctggcaaccg
     2821 tggtgtacaa ccagaaccgt cagcagaacc gtgtgcgcat tttcgaaagc ggtctgcgtt
     2881 tcgtaccaga tactcaggca ccgttgggca ttcgtcagga tctgatgtta gccggtgtga
     2941 tttgcggtaa ccgttacgaa gagcactgga acctggcaaa agagaccgtt gatttctatg
     3001 atttgaaagg cgatcttgaa tccgttctcg acctgaccgg taaactgaat gaggttgagt
     3061 tccgtgcaga agcgaatccg gcactgcatc cggggcaatc cgcagcgatt tatctgaaag
     3121 gtgaacgtat tggttttgtt ggggttgttc atcctgaact ggaacgtaaa ctggatctta
     3181 acggtcgcac tctggtgttc gaactggagt ggaacaagct cgcagaccgc gtggtgcctc
     3241 aggcgcgcga gatttctcgc ttcccggcga accgtcgtga catcgcggtg gtggtcgcag
     3301 aaaacgttcc cgcagcggat attttatccg aatgtaagaa agttggcgta aatcaggtag
     3361 ttggcgtaaa cttatttgac gtgtaccgcg gtaagggtgt tgcggagggg tataagagcc
     3421 tcgccataag cctgatcctg caagatacca gccgtacact cgaagaagag gagattgccg
     3481 ctaccgtcgc caaatgtgta gaggcattaa aagagcgatt ccaggcatca ttgagggatt
     3541 gaaagcttaa ttagctgagc ttggactcct gttgatagat ccagtaatga cctcagaact
     3601 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     3661 aagctagctt ggcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg
     3721 gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt
     3781 cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga
     3841 ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga
     3901 tgaatgctca tccggaattt cgtatggcaa tgaaagacgg tgagctggtg atatgggata
     3961 gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga
     4021 gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt
     4081 acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag
     4141 ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct
     4201 tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc
     4261 tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg
     4321 aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa ggcagttatt
     4381 ggtgccctta aacgcctggg gtaatgactc tctagcttga ggcatcaaat aaaacgaaag
     4441 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     4501 agtaggacaa atccgccctc tagattacgt gcagtcgatg ataagctgtc aaacatgaga
     4561 attgtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4621 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4681 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4741 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     4801 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     4861 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     4921 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     4981 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5041 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5101 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     5161 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5221 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5281 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     5341 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5401 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5461 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     5521 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     5581 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5641 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5701 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg
     5761 gtgtcggaat ttcgggcagc gttgggtcct ggccacgggt gcgcatgatc tagagctgcc
     5821 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     5881 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     5941 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     6001 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     6061 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     6121 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     6181 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     6241 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6301 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6361 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6421 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6481 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6541 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6601 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6661 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6721 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6781 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6841 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6901 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatggcgt taagggattt
     6961 tggtcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     7021 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     7081 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     7141 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     7201 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     7261 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     7321 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     7381 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     7441 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     7501 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     7561 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     7621 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     7681 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     7741 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     7801 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccatga cattaaccta
     7861 taaaaatagg cgtatcacga ggccctttcg tcttcac
//
