LOCUS       sequence-342942-        6292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses T7 RNAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6292)
  AUTHORS   Wimmer F, Englert F, Beisel CL
  TITLE     A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems 
            with Multi-Protein Effector Complexes.
  JOURNAL   Methods Mol Biol. 2022;2433:391-411. doi: 
            10.1007/978-1-0716-1998-8_24.
  PUBMED    34985758
REFERENCE   2  (bases 1 to 6292)
  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 Mol
            Biol."; date: "2022"; volume: "2433:391-411. doi"; pages: " 
            10.1007/978-1-0716-1998-8_24"
COMMENT     Sequence Label: pT7RNAP
FEATURES             Location/Qualifiers
     source          1..6292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             3322..3342
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     rep_origin      3493..3948
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3580..3599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3790..3811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4040..4855)
                     /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(4040..4855)
                     /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     4763..4782
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4847..4866
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4977..5565
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5466..5485
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5719..5736
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5751..5890
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5816..5835)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5976..5998
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5992..6183)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
       61 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
      121 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
      181 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
      241 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
      301 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
      361 gccgccatat gccggcacag aaaagcccgc ctttcggcgg gctttggtcg acggatcaat
      421 tccggaagac gcatctgcca gcctggcaga actgctgaac gcaggtctgg gcggttctga
      481 taacgagtaa tcgttaatcc gcaaataacg taaaaacccg cttcggcggg tttttttatg
      541 gggggagttt agggaaagag catttgtcag aatatttaag gaatttctga atactcataa
      601 tcaatgtaga gatgactaat atcctgaaac tgactgaact aattgagtcg gaattcctcg
      661 agttacgcga acgcgaagtc cgactctaag atgtcacgga ggttcaagtt acctttagcc
      721 ggaagtgctg gcattttgtc caattgagac tcgtgcaact ggtcagcgaa ctggtcgtag
      781 aaatcagcca gtacatcaca agactcatat gtgtcaacca tagtttcgcg cactgctttg
      841 aacaggttcg cagcgtcagc cggaatggta ccgaaggagt cgtgaatcag tgcaaaagat
      901 tcgattccgt acttctcgtg tgcccacact acagtcttac gaaggtggct accgtcttgg
      961 ctgtgtacaa agttaggagc gataccagac tcctgtttgt gtgcatcaat ctcgctatct
     1021 ttgttggtgt taatggtagg ctgtaagcgg aactgaccga ggaacatcag gttcaagcgc
     1081 gtctgaatag gcttcttgta ttcctgccac acagggaaac catcaggagt tacccaatgc
     1141 acagcgcaac gcttgcgaag aatctctcca gtcttcttat ctttgacctc agcagccagc
     1201 agcttagcag cagacttaag ccagttcatt gcttcaaccg cagctaccac cgtcacgctc
     1261 acagattccc aaatcagctt agccatgtat ccagcagcct gattcggctg agtgaacatc
     1321 agacccttgc cggaatcaat agctggctga atggtatctt ccagcacttg ttgacggaag
     1381 ccgaactctt tggacccgta agccagcgtc atgactgaac gcttagtcac actgcgagta
     1441 acaccgtaag ccagccattg accagccagt gccttagtgc ccagcttgac tttctcagag
     1501 atttcaccag tgttctcatc ggtcacggta actacttcgt tatcggtccc attgattgcg
     1561 tctgcttgta gaatctcgtt gactttctta gcaacaatcc cgtagatgtc ctgaacggtt
     1621 tcactaggaa gcaagttaac cgcgcgacca cctacctcat ctcggagcat cgcggagaag
     1681 tgctggatgc cagagcaaga cccgtcaaac gccagcggaa gggagcagtt atagctcagg
     1741 ccgtggtgct gtaccccagc gtactcaaag cagaacgcaa ggaagcagaa cggagaatct
     1801 tgctcagccc accaagtgtt ctccagtgga gacttagcgc aagccatgat gttctcgtgg
     1861 ttttcctcaa tgaacttgat gcgctcaggg aacggaacct tatcgacacc cgcacagttt
     1921 gcaccgtgga ttttcagcca gtagtaacct tccttaccga ttggtttacc tttcgccagc
     1981 gtaagcagtc ctttggtcat atcgttacct tgcgggttga acattgacac agcgtaaaca
     2041 cgaccgcgcc agtccatgtt gtaagggaac cagatggcct tatggttagc aaacttattg
     2101 gcttgctcaa gcatgaactc aaggctgata cggcgagact tgcgagcctt gtccttgcgg
     2161 tacacagcag cggcagcacg tttccacgcg gtgagagcct caggattcat gtcgatgtct
     2221 tccggtttca tcgggagttc ttcacgctca atcgcaggga tgtcctcgac cggacaatgc
     2281 ttccacttgg tgattacgtt ggcgaccgct aggactttct tgttgatttt ccatgcggtg
     2341 ttttgcgcaa tgttaatcgc tttgtacacc tcaggcatgt aaacgtcttc gtagcgcatc
     2401 agtgctttct tactgtgagt acgcaccagc gccagaggac gacgaccgtt agcccaatag
     2461 ccaccaccag taatgccagt ccacggctta ggaggaacta cgcaaggttg gaacatcgga
     2521 gagatgccag ccagcgcacc tgcacgggtt gcgatagcct cagcgtattc aggtgcgagt
     2581 tcgatagtct cagagtcttg acctactacg ccagcatttt ggcggtgtaa gctaaccatt
     2641 ccggttgact caatgagcat ctcgatgcag cgtactccta catgaataga gtcttcctta
     2701 tgccacgaag accacgcctc gccaccgagt agacccttag agagcatgtc agcctcgaca
     2761 acttgcataa atgctttctt gtagacgtgc cctacgcgct tgttgagttg ttcctcaacg
     2821 tttttcttga agtgcttagc ttcaaggtca cggatacgac cgaagcgagc ctcgtcctca
     2881 atggcccgac cgattgcgct tgctacagcc tgaacggttg tattgtcagc actggttagg
     2941 caagccagag tggtcttaat ggtgatgtac gctacggctt ccggcttgat ttcttgcagg
     3001 aactggaagg ctgtcgggcg cttgccgcgc ttagctttca cttcctcaaa ccagtcgttg
     3061 atgcgtgcaa tcatcttagg gagtagggta gtgatgagag gcttggcggc agcgttatcc
     3121 gcaacctcac cagctttaag ttgacgctca aacatcttgc ggaagcgtgc ttcacccatc
     3181 tcgtaagact catgctcaag ggccaactgt tcgcgagcta aacgctcacc gtaatggtca
     3241 gccagagtgt tgaacgggat agcagccagt tcgatgtcag agaagtcgtt cttagcgatg
     3301 ttaatcgtgt tcatttggat cctccttctt aaagttaaac aaaattattg ctagctgcaa
     3361 ccattatcac cgccagaggt aaaattgtca acacgcacgg tgttagctca gcacgttacc
     3421 ggaccagaag ttgtcctggc atgcccagcg gaacagcaca cacatgtaac tatatccgga
     3481 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3541 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3601 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3661 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3721 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3781 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3841 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3901 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     3961 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4021 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     4081 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     4141 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     4201 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     4261 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     4321 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     4381 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     4441 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     4501 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     4561 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     4621 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     4681 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     4741 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     4801 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     4861 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     4921 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4981 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5041 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5101 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5161 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5221 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5281 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5341 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5401 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5461 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5521 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5581 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5641 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     5701 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     5761 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     5821 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     5881 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5941 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     6001 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     6061 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     6121 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     6181 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     6241 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tg
//
