LOCUS       sequence-482091-        9974 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Over-express E. coli Pol III subunit alpha in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9974)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 9974)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Ec dnaE-pET(3c)
FEATURES             Location/Qualifiers
     source          1..9974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     287..306
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     540..557
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    572..711
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(637..656)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     797..819
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(813..1004)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2462..2481
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2565..2584
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2565..2583
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2615..2637
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(8068..8086)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8082..8129
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(8495..8500,8501..8517,8518..8523))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(8550..8568)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8636..8740
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8741..8809,8810..9601)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(8959..8978)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(9772..9974,1..386)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
       61 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
      121 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
      181 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
      241 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
      301 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
      361 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
      421 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
      481 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
      541 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
      601 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
      661 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
      721 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
      781 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
      841 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
      901 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
      961 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     1021 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     1081 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     1141 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     1201 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     1261 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     1321 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     1381 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     1441 aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc
     1501 cgcgtgcggc tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc
     1561 gcattcacag ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta
     1621 gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca
     1681 acgcggggag gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg
     1741 tgctcgccga ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc
     1801 tggtaagagc cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg
     1861 acagcatggc ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg
     1921 ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg
     1981 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     2041 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     2101 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     2161 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     2221 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgacgc tctcccttat
     2281 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     2341 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     2401 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     2461 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     2521 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     2581 agggagacca caacggtttc cctctagaaa taattttgtt taactttaag aaggagatat
     2641 acatatgtct gaaccacgtt tcgtacacct gcgggtgcac agcgactact cgatgatcga
     2701 tggcctggcc aaaaccgcac cgttggtaaa aaaggcggcg gcgttgggta tgccagcact
     2761 ggcgatcacc gatttcacca acctttgtgg tctggtgaag ttctacggag cgggacatgg
     2821 cgcagggatt aagcctatcg tcggggcaga ttttaacgtc cagtgcgacc tgctgggtga
     2881 tgagttaacc cacctgacgg tactggcggc gaacaatacc ggctatcaga atctgacgtt
     2941 gctgatctca aaagcgtatc agcgcgggta cggtgccgcc gggccgatca tcgatcgcga
     3001 ctggcttatc gaattaaacg aagggttgat ccttctttcc ggcggacgca tgggcgacgt
     3061 cggacgcagt cttttgcgtg gtaacagcgc gctggtagat gagtgtgtcg cgttttatga
     3121 agaacacttc ccggatcgct attttctcga gctgatccgc accggcaggc cggatgaaga
     3181 aagctatctg cacgcggcgg tggaactggc ggaagcgcgc ggtttgcccg tcgtggcgac
     3241 caacgacgtg cgctttatcg acagcagcga ctttgacgca cacgaaatcc gcgtcgcgat
     3301 ccacgacggc tttaccctcg acgatcctaa acgcccgcgt aactattcgc cgcagcaata
     3361 tatgcgtagc gaagaggaga tgtgtgagct gtttgccgac atccccgaag cccttgccaa
     3421 caccgttgag atcgccaaac gctgtaacgt aaccgtgcgt cttggtgaat acttcctgcc
     3481 gcagttcccg accggggaca tgagcaccga agattatctg gtcaagcgtg caaaagaggg
     3541 cctggaagag cgtctggcct ttttattccc tgatgaggaa gaacgtctta agcgccgccc
     3601 ggaatatgac gaacgtctgg agactgaact tcaggttatc aaccagatgg gcttcccggg
     3661 ctacttcctc atcgttatgg aatttatcca gtggtcgaaa gataacggcg taccggtagg
     3721 gccaggccgt ggctccggtg cgggttcact ggtggcctac gcgctgaaaa tcaccgacct
     3781 cgatccgctg gaatttgacc tgctgttcga acgtttcctt aacccggaac gtgtctccat
     3841 gcctgacttc gacgttgact tctgtatgga gaaacgcgat caggttatcg agcacgtagc
     3901 ggacatgtac ggtcgtgatg cggtatcgca gatcatcacc ttcggtacaa tggcggcgaa
     3961 agcggtgatc cgcgacgtag gccgcgtgct ggggcatccg tacggctttg tcgatcgtat
     4021 ctcgaaactg atcccgcccg atccggggat gacgctggcg aaagcgtttg aagccgagcc
     4081 gcagctgccg gaaatctacg aagcggatga agaagttaag gcgctgatcg acatggcgcg
     4141 caaactggaa ggggtcaccc gtaacgccgg taagcacgcc ggtggggtgg ttatcgcgcc
     4201 gaccaaaatt accgattttg cgccgcttta ctgcgatgaa gagggcaaac atccggtcac
     4261 ccagtttgat aaaagcgacg ttgaatacgc cggactggtg aagttcgact tccttggttt
     4321 gcgtacgctc accatcatca actgggcgct ggagatgatc aacaagcggc gggcgaagaa
     4381 tggcgagccg ccgctggata tcgctgcgat cccgctggat gataagaaaa gcttcgacat
     4441 gctgcaacgc tcggaaacca cggcggtatt ccagcttgaa tcgcgcggca tgaaggacct
     4501 gatcaagcgt ctacaacctg actgcttcga agatatgatc gccctagtgg cactgttccg
     4561 ccccggtccg ttgcaatcag ggatggtgga taactttatc gaccgtaaac atggtcgtga
     4621 agagatctcc tatccggacg tacagtggca gcatgaaagc ctgaaaccgg tactggagcc
     4681 aacctacggc attatcctgt atcaggaaca ggtcatgcag attgcgcagg tgctttctgg
     4741 ttataccctc ggtggcgcgg atatgctgcg tcgtgcgatg ggtaagaaaa agccggaaga
     4801 gatggctaag caacgttctg tatttgctga aggtgcagaa aagaacggaa tcaacgctga
     4861 actggcgatg aaaatcttcg acctggtgga gaaattcgct ggttacggat ttaacaaatc
     4921 gcactctgcg gcctatgctt tggtgtcata tcaaacgtta tggctgaaag cgcactatcc
     4981 tgcggagttt atggcggcgg taatgaccgc cgatatggac aacaccgaga aggtggtggg
     5041 tctggtggat gagtgctggc ggatggggct gaaaatcctg ccaccagata taaactccgg
     5101 tctttaccat ttccacgtca acgacgacgg cgaaatcgtg tatggtattg gcgcgatcaa
     5161 aggggtcggt gaaggtccga ttgaggccat catcgaagcc cgtaataaag gcggctactt
     5221 ccgcgaactg tttgatctct gcgcccgtac cgacaccaaa aagttgaacc gtcgcgtgct
     5281 ggaaaaactg atcatgtccg gggcgtttga ccgtcttggg ccacatcgcg cagcgctgat
     5341 gaactcgctg ggcgatgcgt taaaagcggc agatcaacac gcgaaagcgg aagctatcgg
     5401 tcaggccgat atgttcggcg tgctggccga agagccggaa caaattgaac aatcctacgc
     5461 cagctgccaa ccgtggccgg agcaggtggt attagatggg gaacgtgaaa cgttaggcct
     5521 gtacctgacc ggacacccta tcaaccagta tttaaaagag attgagcgtt atgtcggagg
     5581 cgtaaggctg aaagacatgc acccgacaga acgtggtaaa gtcatcacgg ctgcggggct
     5641 cgttgttgcc gcgcgggtta tggtcaccaa gcgcggcaat cgtatcggta tctgcacgct
     5701 ggatgaccgt tccgggcggc tggaagtgat gttgtttact gacgccctgg ataaatacca
     5761 gcaattgctg gaaaaagacc gcatacttat cgtcagcgga caggtcagct ttgatgactt
     5821 cagcggtggg cttaaaatga ccgctcgcga agtgatggat attgacgaag cccgggaaaa
     5881 atatgctcgc gggcttgcta tctcgctgac ggacaggcaa attgatgacc agcttttaaa
     5941 ccgactccgt cagtctctgg aaccccaccg ctctgggaca attccagtac atctctacta
     6001 tcagagggcg gatgcacgcg cgcggttgcg ttttggcgcg acgtggcgtg tctctccgag
     6061 cgatcgttta ttaaacgatc tccgtggcct cattggttcg gagcaggtgg aactggagtt
     6121 tgactaatac aggaatacta tgagtctgaa tttccttgat tttgaacagc cgattgcaga
     6181 gctggaagcg aaaatcgatt ctctgactgc ggttagccgt caggatgaga aactggatat
     6241 taacatcgat gaagaagtgc atcgtctgcg tgaaaaaagc gtagaactga cacgtaaaat
     6301 cttcgccgat ctcggtgcat ggcagattgc gcaactggca cgccatccac agcgtcctta
     6361 taccctggat tacgttcgcc tggcatttga tgaatttgac gaactggctg gcgaccgcgc
     6421 gtatgcagac gataaagcta tcgtcggtgg tatcgcccgt ctcgatggtc gtccggtgat
     6481 gatcattggt catcaaaaag gtcgtgaaac caaagaaaaa attcgccgta actttggtat
     6541 gccagcgcca gaaggttacc gcaaagcact gcgtctgatg caaatggctg aacgctttaa
     6601 gatgcctatc atcaccttta tcgacacccc gggggcttat cctggcgtgg gcgcagaaga
     6661 gcgtggtcag tctgaagcca ttgcacgcaa cctgcgtgaa atgtctcgcc tcggcgtacc
     6721 ggtagtttgt acggttatcg gtgaaggtgg ttctggcggt gcgctggcga ttggcgtggg
     6781 cgataaagtg aatatgctgc aatacagcac ctattccgtt atctcgccgg aaggttgtgc
     6841 gtccattctg tggaagagcg ccgacaaagc gccgctggcg gctgaagcga tgggtatcat
     6901 tgctccgcgt ctgaaagaac tgaaactgat cgactccatc atcccggaac cactgggtgg
     6961 tgctcaccgt aacccggaag cgatggcggc atcgttgaaa gcgcaactgc tggcggatct
     7021 ggccgatctc gacgtgttaa gcactgaaga tttaaaaaat cgtcgttatc agcgcctgat
     7081 gagctacggt tacgcgtaat tcgcaaaagt tctgaaaaag ggtcacttcg gtggcccttt
     7141 tttatcgcca cggtttgagc aggctatgat taaggaagga ttttccagga ggaacacatg
     7201 aacatcattg ccattatggg accgcatggc gtcttttata aagatgagcc catcaaagaa
     7261 ctggagtcgg cgctggtggc gcaaggcttt cagattatct ggccacaaaa cagcgttgat
     7321 ttgctgaaat ttatcgagca taaccctcga atttgcggcg tgatttttga ctgggatgag
     7381 tacagtctcg atttatgtag cgatatcaat cagcttaatg aatatctccc gctttatgcc
     7441 ttcatcaaca cccactcgac gatggatgtc agcgtgcagg atatgcggat ggcgctctgg
     7501 ttttttgaat atgcgctggg gcaggcggaa gatatcgcca ttcgtatgcg tcagtacacc
     7561 gacgaatatc ttgataacat tacaccgccg ttcacgaaag ccttgtttac ctacgtcaaa
     7621 gagcggaagt acaccttttg tacgccgggg catatgggcg gcaccgcata tcaaaaaagc
     7681 ccggttggct gtctgtttta tgattttttc ggcgggaata ctcttaaggc tgatgtctct
     7741 atttcggtca ccgagcttgg ttcgttgctc gaccacaccg ggccacacct ggaagcggaa
     7801 gagtacatcg cgcggacttt tggcgcggaa cagagttata tcgttaccaa cggaacatcg
     7861 acgtcgaaca aaattgtggg tatgtacgcc gcgccatccg gcagtacgct gttgatcgac
     7921 cgcaattgtc ataaatcgct ggcgcatctg ttgatgatga acgatgtagt gccagtctgg
     7981 ctgaaaccga cgcgtaatgc gttggggatt cttggtggga tccggctgct aacaaagccc
     8041 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     8101 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatccac
     8161 aggacgggtg tggtcgccat gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc
     8221 aggactgggc ggcggccaaa gcggtcggac agtgctccga gaacgggtgc gcatagaaat
     8281 tgcatcaacg catatagcgc tagcagcacg ccatagtgac tggcgatgct gtcggaatgg
     8341 acgatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc
     8401 agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga
     8461 ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt
     8521 caaacatgag aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     8581 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     8641 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     8701 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     8761 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     8821 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     8881 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     8941 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     9001 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     9061 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     9121 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     9181 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     9241 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     9301 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     9361 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     9421 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     9481 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     9541 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     9601 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     9661 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     9721 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     9781 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     9841 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     9901 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     9961 tgtagcaccg ccta
//
