LOCUS       sequence-419578-        5428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a T7 RNA Polymerase that produces long RNA transcripts in 
            high yield and generates fewer abortive transcripts..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5428)
  AUTHORS   Chillon I, Marcia M, Legiewicz M, Liu F, Somarowthu S, Pyle AM
  TITLE     Native Purification and Analysis of Long RNAs.
  JOURNAL   Methods Enzymol. 2015;558:3-37. doi: 10.1016/bs.mie.2015.01.008. 
            Epub 2015 Feb 27.
  PUBMED    26068736
REFERENCE   2  (bases 1 to 5428)
  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: "Methods 
            Enzymol."; date: "2015"; volume: "558:3-37. doi"; pages: " 
            10.1016/bs.mie.2015.01.008. Epub 2015 Feb 27"
COMMENT     Sequence Label: p6XHis-T7(P266L)
FEATURES             Location/Qualifiers
     source          1..5428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(673..678,679..695,696..701))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(728..746)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        814..918
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(919..987,988..1779)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1137..1156)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1950..2538
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2439..2458
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2692..2709
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2826..2847
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2862..2867,2868..2885,2886..2892)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2900..2916
                     /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     2905..2927
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2924..2940
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2924..2940
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     RBS             2989..3011
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3030..3047
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3057..3074
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 ctttgtacac agccaagacg gtagccacct tcgtaagact gtagtgtggg cacacgagaa
       61 gtacggaatc gaatcttttg cactgattca cgactccttc ggtaccattc cggctgacgc
      121 tgcgaacctg ttcaaagcag tgcgcgaaac tatggttgac acatatgagt cttgtgatgt
      181 actggctgat ttctacgacc agttcgctga ccagttgcac gagtctcaat tggacaaaat
      241 gccagcactt ccggctaaag gtaacttgaa cctccgtgac atcttagagt cggacttcgc
      301 gttcgcgtaa cgccaaatca atacgactcc ggatccacag gacgggtgtg gtcgccatga
      361 tcgcgtagtc gatagtggct ccaagtagcg aagcgagcag gactgggcgg cggccaaagc
      421 ggtcggacag tgctccgaga acgggtgcgc atagaaattg catcaacgca tatagcgcta
      481 gcagcacgcc atagtgactg gcgatgctgt cggaatggac gatatcccgc aagaggcccg
      541 gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga
      601 cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg
      661 ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga
      721 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
      781 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
      841 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
      901 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
      961 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1021 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1081 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     1141 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     1201 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     1261 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     1321 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     1381 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     1441 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     1501 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     1561 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     1621 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     1681 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     1741 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     1801 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     1861 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1921 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgtg cgtaatctgc
     1981 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2041 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     2101 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2161 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2221 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2281 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2341 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2401 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2461 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2521 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2581 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2641 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2701 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     2761 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     2821 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     2881 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     2941 catgattacg ccaagcttgc atgcctgcag gtcgactcta gaaataattt tgtttaactt
     3001 taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc agcggcctgg
     3061 tgccgcgcgg cagccatatg ctcgagatga acacgattaa catcgctaag aacgacttct
     3121 ctgacatcga actggctgct atcccgttca acactctggc tgaccattac ggtgagcgtt
     3181 tagctcgcga acagttggcc cttgagcatg agtcttacga gatgggtgaa gcacgcttcc
     3241 gcaagatgtt tgagcgtcaa cttaaagctg gtgaggttgc ggataacgct gccgccaagc
     3301 ctctcatcac taccctactc cctaagatga ttgcacgcat caacgactgg tttgaggaag
     3361 tgaaagctaa gcgcggcaag cgcccgacag ccttccagtt cctgcaagaa atcaagccgg
     3421 aagccgtagc gtacatcacc attaagacca ctctggcttg cctaaccagt gctgacaata
     3481 caaccgttca ggctgtagca agcgcaatcg gtcgggccat tgaggacgag gctcgcttcg
     3541 gtcgtatccg tgaccttgaa gctaagcact tcaagaaaaa cgttgaggaa caactcaaca
     3601 agcgcgtagg gcacgtctac aagaaagcat ttatgcaagt tgtcgaggct gacatgctct
     3661 ctaagggtct actcggtggc gaggcgtggt cttcgtggca taaggaagac tctattcatg
     3721 taggagtacg ctgcatcgag atgctcattg agtcaaccgg aatggttagc ttacaccgcc
     3781 aaaatgctgg cgtagtaggt caagactctg agactatcga actcgcacct gaatacgctg
     3841 aggctatcgc aacccgtgca ggtgcgctgg ctggcatctc tctgatgttc caaccttgcg
     3901 tagttcctcc taagccgtgg actggcatta ctggtggtgg ctattgggct aacggtcgtc
     3961 gtcctctggc gctggtgcgt actcacagta agaaagcact gatgcgctac gaagacgttt
     4021 acatgcctga ggtgtacaaa gcgattaaca ttgcgcaaaa caccgcatgg aaaatcaaca
     4081 agaaagtcct agcggtcgcc aacgtaatca ccaagtggaa gcattgtccg gtcgaggaca
     4141 tccctgcgat tgagcgtgaa gaactcccga tgaaaccgga agacatcgac atgaatcctg
     4201 aggctctcac cgcgtggaaa cgtgctgccg ctgctgtgta ccgcaaggac aaggctcgca
     4261 agtctcgccg tatcagcctt gagttcatgc ttgagcaagc caataagttt gctaaccata
     4321 aggccatctg gttcccttac aacatggact ggcgcggtcg tgtttacgct gtgtcaatgt
     4381 tcaacccgca aggtaacgat atgaccaaag gactgcttac gctggcgaaa ggtaaaccaa
     4441 tcggtaagga aggttactac tggctgaaaa tccacggtgc aaactgtgcg ggtgtcgata
     4501 aggttccgtt ccctgagcgc atcaagttca ttgaggaaaa ccacgagaac atcatggctt
     4561 gcgctaagtc tccactggag aacacttggt gggctgagca agattctccg ttctgcttcc
     4621 ttgcgttctg ctttgagtac gctggggtac agcaccacgg cctgagctat aactgctccc
     4681 ttccgctggc gtttgacggg tcttgctctg gcatccagca cttctccgcg atgctccgag
     4741 atgaggtagg tggtcgcgcg gttaacttgc ttcctagtga aaccgttcag gacatctacg
     4801 ggattgttgc taagaaagtc aacgagattc tacaagcaga cgcaatcaat gggaccgata
     4861 acgaagtagt taccgtgacc gatgagaaca ctggtgaaat ctctgagaaa gtcaagctgg
     4921 gcactaaggc actggctggt caatggctgg cttacggtgt tactcgcagt gtgactaagc
     4981 gttcagtcat gacgctggct tacgggtcca aagagttcgg cttccgtcaa caagtgctgg
     5041 aagataccat tcagccagct attgattccg gcaagggtct gatgttcact cagccgaatc
     5101 aggctgctgg atacatggct aagctgattt gggaatctgt gagcgtgacg gtggtagctg
     5161 cggttgaagc aatgaactgg cttaagtctg ctgctaagct gctggctgct gaggtcaaag
     5221 ataagaagac tggagagatt cttcgcaagc gttgcgctgt gcattgggta actcctgatg
     5281 gtttccctgt gtggcaggaa tacaagaagc ctattcagac gcgcttgaac ctgatgttcc
     5341 tcggtcagtt ccgcttacag cctaccatta acaccaacaa agatagcgag attgatgcac
     5401 acaaacagga gtctggtatc gctcctaa
//
