LOCUS       sequence-429515-        6670 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express T. aq sigmaA transcription initiation factor in E. coli 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6670)
  AUTHORS   Hertz LM, White EN, Kuznedelov K, Cheng L, Yu AM, Kakkaramadam R, 
            Severinov K, Chen A, Lucks JB
  TITLE     The effect of pseudoknot base pairing on cotranscriptional 
            structural switching of the fluoride riboswitch.
  JOURNAL   Nucleic Acids Res. 2024 May 8;52(8):4466-4482. doi: 
            10.1093/nar/gkae231.
  PUBMED    38567721
REFERENCE   2  (bases 1 to 6670)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2024-05-8- 8"; volume: "52"; issue: "8";
            pages: "4466-4482"
COMMENT     Sequence Label: T. aq sigmaA
FEATURES             Location/Qualifiers
     source          1..6670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(325..342)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(352..369)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             388..410
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    425..449
                     /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     complement(449..468)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(450..468)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(552..571)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        777..854
                     /gene="lacI"
                     /label=lacI promoter
     CDS             855..1937
                     /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     complement(874..893)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2746..2937
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2931..2953)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3039..3178
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3094..3113
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3193..3210)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3363..3951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3443..3462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4062..4081)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4073..4888
                     /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             4073..4888
                     /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     complement(4146..4165)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4980..5435)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5117..5138)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5329..5348
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5472..5519)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5515..5533
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5586..5603)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gggcacctgg ccgatctcgt ggaggtactg gcgcacgggg tcggaggtgg agaccttggg
       61 gagggaaagc tcctcctctt cctcctccaa aagcccctcc tcgggcagga gatcggcctc
      121 cagggggccc tcgaggtcca aaggatcggc caggtcctcc agctcgggct ccgaggccag
      181 gagctcgggg tcgggctcgg ggaggtcctg gaggtcctcg gccgcctcca gctcggggag
      241 gggctcgggc tccttgacgg gttccttcac ctccacctcc tgggccttgg ccgccttctt
      301 cttgctcttg gactttttca tatggctgcc gcgcggcacc aggccgctgc tgtgatgatg
      361 atgatgatgg ctgctgccca tggtatatct ccttcttaaa gttaaacaaa attatttcta
      421 gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat
      481 cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
      541 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
      601 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
      661 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
      721 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
      781 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
      841 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
      901 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
      961 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     1021 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     1081 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     1141 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     1201 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     1261 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     1321 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     1381 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     1441 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     1501 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     1561 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     1621 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     1681 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     1741 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     1801 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     1861 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     1921 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     1981 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     2041 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
     2101 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
     2161 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
     2221 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca
     2281 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     2341 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     2401 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     2461 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     2521 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     2581 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     2641 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     2701 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     2761 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     2821 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     2881 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     2941 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     3001 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     3061 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     3121 tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     3181 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3241 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3301 gaacatgtga gcaaaaggcc agcaaaaggc caggaccgta aaaaggccgc gttgctggcg
     3361 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3421 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3481 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3541 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3601 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3661 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3721 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3781 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     3841 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3901 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3961 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4021 gtcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     4081 tattcaacgg gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt
     4141 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt
     4201 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     4261 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     4321 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     4381 gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     4441 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     4501 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     4561 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     4621 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     4681 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     4741 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     4801 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     4861 tcatttgatg ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat
     4921 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     4981 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     5041 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5101 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5161 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     5221 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5281 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5341 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5401 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccaatcc ggatatagtt
     5461 cctcctttca gcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctagt
     5521 tattgctcag cggtggcagc agccaactca gcttcctttc gggctttgtt agcagccgga
     5581 tctcagtggt ggtggtggtg gtgctcgagt gcggccgcaa gcttgtcgac ggagctcgaa
     5641 ttcgagctcg gtacccgggg atccgatgcg gatcctattc caagaagtcc cggagcttgc
     5701 gggtgcggga ctcgtggtac ttgagcttcc ttaaggcctt gttctcaatc tggcggatgc
     5761 gctcccgggt cacgccaaag taggccccca cctcctcgag ggtgtgctcc cggccgtcaa
     5821 tgagcccttt ccggagcttc agcaccatgg cttcccgctc ggagagcttg gaaagggcct
     5881 tctccagctc ctcggagagg aggctctggg ccgccgcctc cacgggggag ggcaggttct
     5941 cgtcggggat gaagtcgccg tagaagctgt ccttctcgtc gccgatgggg gtttccaggg
     6001 agacgggctc ctgggcgatc tttagggtct cctccacccg cttggcgtcc cagccggggc
     6061 ccatggcctc ggcgatctcc tcgtaggagg gctcccggcc cagctcctgc tggagctgtc
     6121 tggcggtgcg ggagagcttg ttgatggtct ccaccatgtg gaccgggatg cggatggtgc
     6181 gggcctggtc ggcgatggcc cggttgatgg cctggcggat ccaccaggtg gcgtaggtgg
     6241 agaacttgaa gcggcgcttg tactcaaact tctccaccgc ccggatgagg ccctggttgc
     6301 cctcctggat gaggtccagg aagctcaggc cccgaccggt gtacttcttg gcgatggaga
     6361 ccacgagcct gaggttggcc tcaatgaggt gctgcctggc cgcctccccc tcccgggcga
     6421 tgtgcaggta gcgcttgagc tccttgggga ggcttttcag cttcccgtcc acctcctcaa
     6481 ccgtcttggg gtccggcttc tccttgaggc cggggatctt ttggatccgg gccgtgccca
     6541 ggatcttggc ccgcaccacc tcgcggatga gttcctggtc taggcccgtg gcctcggaga
     6601 gcttcttgat ggcctccatg ccctcctcca ccttcctggc caggtcaatc tcctcttcca
     6661 gggtgaggag
//
