LOCUS       sequence-197800-        7154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses T7 RNA polymerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7154)
  AUTHORS   Abil Z, Ellefson JW, Gollihar JD, Watkins E, Ellington AD
  TITLE     Compartmentalized partnered replication for the directed evolution 
            of genetic parts and circuits.
  JOURNAL   Nat Protoc. 2017 Dec;12(12):2493-2512. doi: 10.1038/nprot.2017.119. 
            Epub 2017 Nov 9.
  PUBMED    29120463
REFERENCE   2  (bases 1 to 7154)
  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"; doi: 
            "10.1038/nprot.2017.119"; journalName: "Nat Protoc"; date: 
            "2017-12"; volume: "12"; issue: "12"; pages: "2493-2512"
COMMENT     Sequence Label: pQE 80L Kan T7WT
FEATURES             Location/Qualifiers
     source          1..7154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     968..987
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1007..1084)
                     /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(1168..1190)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1276..1416
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1331..1350
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1431..1448)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1602..2190)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1682..1701)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2297..3112)
                     /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(2297..3112)
                     /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     3020..3039
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3104..3123
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3184..3202
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3226..3240,3241..3246,3247..3263,3264..3270)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3246..3262
                     /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    3278..3294
                     /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             3313..3324
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3343..3360
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      6037..6131
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             6175..6834
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(6241..6260)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6899..6985
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(7078..7154,1..1006))
                     /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"
ORIGIN
        1 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
       61 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
      121 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
      181 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
      241 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
      301 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
      361 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
      421 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
      481 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
      541 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
      601 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
      661 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
      721 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
      781 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
      841 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
      901 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
      961 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     1021 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac cattcgatgg
     1081 tgtcggaatt tcgggcagcg ttgggtcctg gccacgggtg cgcatgatct agagctgcct
     1141 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     1201 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     1261 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     1321 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     1381 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     1441 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     1501 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     1561 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1621 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1681 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1741 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1801 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1861 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1921 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1981 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2041 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2101 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2161 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2221 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatggcgtt aagggatttt
     2281 ggtcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2341 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2401 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     2461 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2521 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2581 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2641 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     2701 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2761 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2821 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2881 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2941 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3001 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3061 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3121 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccatgac attaacctat
     3181 aaaaataggc gtatcacgag gccctttcgt cttcacctcg agaaatcata aaaaatttat
     3241 ttgctttgtg agcggataac aattataata gattcaattg tgagcggata acaatttcac
     3301 acagaattca ttaaagagga gaaattaact atgagaggat cgcatcacca tcaccatcac
     3361 ggatccaaca cgattaacat cgctaagaac gacttctctg acatcgaact ggctgctatc
     3421 ccgttcaaca ctctggctga ccattacggt gagcgtttag ctcgcgaaca gttggccctt
     3481 gagcatgagt cttacgagat gggtgaagca cgcttccgca agatgtttga gcgtcaactt
     3541 aaagctggtg aggttgcgga taacgctgcc gccaagcctc tcatcactac cctactccct
     3601 aagatgattg cacgcatcaa cgactggttt gaggaagtga aagctaagcg cggcaagcgc
     3661 ccgacagcct tccagttcct gcaagaaatc aagccggaag ccgtagcgta catcaccatt
     3721 aagaccactc tggcttgcct aaccagtgct gacaatacaa ccgttcaggc tgtagcaagc
     3781 gcaatcggtc gggccattga ggacgaggct cgcttcggtc gtatccgtga ccttgaagct
     3841 aagcacttca agaaaaacgt tgaggaacaa ctcaacaagc gcgtagggca cgtctacaag
     3901 aaagcattta tgcaagttgt cgaggctgac atgctctcta agggtctact cggtggcgag
     3961 gcgtggtctt cgtggcataa ggaagactct attcatgtag gagtacgctg catcgagatg
     4021 ctcattgagt caaccggaat ggttagctta caccgccaaa atgctggcgt agtaggtcaa
     4081 gactctgaga ctatcgaact cgcacctgaa tacgctgagg ctatcgcaac ccgtgcaggt
     4141 gcgctggctg gcatctctcc gatgttccaa ccttgcgtag ttcctcctaa gccgtggact
     4201 ggcattactg gtggtggcta ttgggctaac ggtcgtcgtc ctctggcgct ggtgcgtact
     4261 cacagtaaga aagcactgat gcgctacgaa gacgtttaca tgcctgaggt gtacaaagcg
     4321 attaacattg cgcaaaacac cgcatggaaa atcaacaaga aagtcctagc ggtcgccaac
     4381 gtaatcacca agtggaagca ttgtccggtc gaggacatcc ctgcgattga gcgtgaagaa
     4441 ctcccgatga aaccggaaga catcgacatg aatcctgagg ctctcaccgc gtggaaacgt
     4501 gctgccgctg ctgtgtaccg caaggacaag gctcgcaagt ctcgccgtat cagccttgag
     4561 ttcatgcttg agcaagccaa taagtttgct aaccataagg ccatctggtt cccttacaac
     4621 atggactggc gcggtcgtgt ttacgctgtg tcaatgttca acccgcaagg taacgatatg
     4681 accaaaggac tgcttacgct ggcgaaaggt aaaccaatcg gtaaggaagg ttactactgg
     4741 ctgaaaatcc acggtgcaaa ctgtgcgggt gtcgataagg ttccgttccc tgagcgcatc
     4801 aagttcattg aggaaaacca cgagaacatc atggcttgcg ctaagtctcc actggagaac
     4861 acttggtggg ctgagcaaga ttctccgttc tgcttccttg cattctgctt tgagtacgct
     4921 ggggtacagc accacggcct gagctataac tgctcccttc cgctggcgtt tgacgggtct
     4981 tgctctggca tccagcactt ctccgcgatg ctccgagatg aggtaggtgg tcgcgcggtt
     5041 aacttgcttc ctagtgaaac cgttcaggac atctacggga ttgttgctaa gaaagtcaac
     5101 gagattctac aagcagacgc aatcaatggg accgataacg aagtagttac cgtgaccgat
     5161 gagaacactg gtgaaatctc tgagaaagtc aagctgggca ctaaggcact ggctggtcaa
     5221 tggctggctt acggtgttac tcgcagtgtg actaagcgtt cagtcatgac gctggcttac
     5281 gggtccaaag agttcggctt ccgtcaacaa gtgctggaag ataccattca gccagctatt
     5341 gattccggca agggtctgat gttcactcag ccgaatcagg ctgctggata catggctaag
     5401 ctgatttggg aatctgtgag cgtgacggtg gtagctgcgg ttgaagcaat gaactggctt
     5461 aagtctgctg ctaagctgct ggctgctgag gtcaaagata agaagactgg agagattctt
     5521 cgcaagcgtt gcgctgtgca ttgggtaact cctgatggtt tccctgtgtg gcaggaatac
     5581 aagaagccta ttcagacgcg cttgaacctg atgttcctcg gtcagttccg cttacagcct
     5641 accattaaca ccaacaaaga tagcgagatt gatgcacaca aacaggagtc tggtatcgct
     5701 cctaactttg tacacagcca agacggtagc caccttcgta agactgtagt gtgggcacac
     5761 gagaagtacg gaatcgaatc ttttgcactg attcacgact ccttcggtac cattccggct
     5821 gacgctgcga acctgttcaa agcagtgcgc gaaactatgg ttgacacata tgagtcttgt
     5881 gatgtactgg ctgatttcta cgaccagttc gctgaccagt tgcacgagtc tcaattggac
     5941 aaaatgccag cacttccggc taaaggtaac ttgaacctcc gtgacatctt agagtcggac
     6001 ttcgcgttcg cgtaaaagct taattagctg agcttggact cctgttgata gatccagtaa
     6061 tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta
     6121 ttggtgagaa tccaagctag cttggcgaga ttttcaggag ctaaggaagc taaaatggag
     6181 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     6241 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     6301 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     6361 cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga cggtgagctg
     6421 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     6481 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     6541 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     6601 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     6661 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     6721 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     6781 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt
     6841 taaggcagtt attggtgccc ttaaacgcct ggggtaatga ctctctagct tgaggcatca
     6901 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     6961 gaacgctctc ctgagtagga caaatccgcc ctctagatta cgtgcagtcg atgataagct
     7021 gtcaaacatg agaattgtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     7081 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     7141 gcggggagag gcgg
//
