LOCUS       sequence-482110-        7139 bp    DNA     circular SYN 06-AUG-2025
DEFINITION  Over-express Ec FLAG-dnaB subunit in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7139)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 7139)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Ec FLAG-dnaB-pET
FEATURES             Location/Qualifiers
     source          1..7139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(162..750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(242..261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(921..1712,1713..1781))
                     /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     1544..1563
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1782..1886)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1954..1972
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1999..2004,2005..2021,2022..2027)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(2202..2249)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2245..2263
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3748..3813)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     RBS             3823..3845
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3860..3884
                     /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(3884..3903)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3885..3903)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3987..4006)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4212..4289
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(4309..4328)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6683..6874
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6868..6890)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6976..7115
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     7031..7050
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(join(7130..7139,1..8))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
       61 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      121 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      181 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      241 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      301 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      361 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      421 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      481 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      541 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      601 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
      661 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
      721 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
      781 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
      841 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
      901 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
      961 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1021 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     1081 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     1141 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     1201 ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg
     1261 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     1321 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     1381 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     1441 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     1501 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat
     1561 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     1621 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     1681 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     1741 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     1801 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     1861 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     1921 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     1981 cttcaagaat tctcatgttt gacagcttat catcgataag ctttaatgcg gtagtttatc
     2041 acagttaaat tgctaacgca gtcaggcacc gtgtatgaaa tctaacaatg cgctcatcgt
     2101 catcctcggc accgtcaccc tggatgctgt aggcataggc ttggttatgc cggtactgcc
     2161 gggcctcttg cgggatatcc ggatatagtt cctcctttca gcaaaaaacc cctcaagacc
     2221 cgtttagagg ccccaagggg ttatgctagt tattgctcag cggtggcagc agccaactca
     2281 gcttcctttc gggctttgtt agcagccgga tccttattat tcgtcgtcgt actgcggccc
     2341 cgcatagttg tcgaagcgcg accattgacc gttaaaggtc aggcgtaccg tcccgattgg
     2401 gccgttacgt tgtttaccga taataatttc cgcgatgcct tttaaatcac tgttttcgtg
     2461 atacacctca tcacgataga taaacatgat caagtccgca tcctgctcga tagagccaga
     2521 ttcacgcagg tcggagttga ccgggcgttt gtcggcacgt tgttccagag aacggttcaa
     2581 ctgggacagc gccaccaccg gcacgttcag ttctttcgcc agtgctttca gcgagcgaga
     2641 gatttctgca atttccagcg tacggttatc ggaaagcgcc ggtacgcgca tcagttgcag
     2701 gtagtcgatc atgataagcc cgatgccgcc gtgttcacgg gcaatacggc gtgcgcggga
     2761 acgcacttcc gttggcgtca ggccggagga gtcatcgata tagatattgc gtttttcgag
     2821 caaaataccc atggtgccgg aaatgcgcgc ccagtcttcg tcatcgagct gcccggtacg
     2881 gattttagtc tggtcaacgc gcgacagcga cgccagagaa cgcatcatga tctgttctga
     2941 tggcatctcc agcgagaaga taagtaccgg tttatcctgc aacatcgccg cgttttcgac
     3001 gaggttcatc gcaaatgttg ttttacccat cgacggacgc gcggcgacga tgatcaaatc
     3061 cgacggctgc aagccagcgg tttttttgtt gagatcgtca taaccggtgt ttaccccggt
     3121 aacgccatcg tgtggctgct gaaacaactg ctcaatacgc gccacggttg cgtcgagcac
     3181 atcggcgatg ttcttcggcc cttcgtcttt gttcgcacga ctttcggcaa ttttaaagac
     3241 gcgggattca gccagatcca gcagatcttc gctggtacgc ccctgcggat caaaaccagc
     3301 ttcggcaatc tcattcgcaa ccgagatcat ctcacggaca acggcacgtt cacgcacgat
     3361 gtccgcatag gcactgatgt tagccgcact tggcgtattt tttgacagct ctgccagata
     3421 agcaaaacca ccgacgctat cgagttgccc ctggcgttcc agcgattccg caagagtaat
     3481 cagatcgata gggctaccgc tttcctgcaa acgcgccatt tcagtaaaga tatgacggtg
     3541 tgggcgggtg taaaaatcgt ctgctaccac acgctcggct acatcatccc agcgttcgtt
     3601 atctagcatt aaaccgccca acaccgactg ctccgcttcg atcgagtgcg gaggcacttt
     3661 cagcccggca acttgtggat cgcgttcgcg gggttcagcc tgctgtttgt tgaagggttt
     3721 atttcctgcc atatgactag tggatcgctt gtcatcgtca tccttgtagt cgatgtcatg
     3781 atctttataa tcaccgtcat ggtctttgta gtccatggta tatctccttc ttaaagttaa
     3841 acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta
     3901 ttaatttcgc gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac
     3961 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
     4021 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
     4081 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
     4141 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     4201 tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     4261 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     4321 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     4381 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     4441 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     4501 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     4561 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     4621 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     4681 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     4741 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     4801 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     4861 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     4921 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     4981 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     5041 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     5101 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     5161 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     5221 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     5281 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     5341 gacagtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     5401 ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
     5461 tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc
     5521 aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct
     5581 ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc
     5641 aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg
     5701 gcatggcggc cgacgcgctg ggctacgtct tgctggcgtt cgcgacgcga ggctggatgg
     5761 ccttccccat tatgattctt ctcgcttccg gcggcatcgg gatgcccgcg ttgcaggcca
     5821 tgctgtccag gcaggtagat gacgaccatc agggacagct tcaaggatcg ctcgcggctc
     5881 ttaccagcct aacttcgatc attggaccgc tgatcgtcac ggcgatttat gccgcctcgg
     5941 cgagcacatg gaacgggttg gcatggattg taggcgccgc cctatacctt gtctgcctcc
     6001 ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gccggcggca
     6061 cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt
     6121 gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg
     6181 cacgcggcgc atctcgggca gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct
     6241 gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc
     6301 gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac
     6361 atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg
     6421 caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac
     6481 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     6541 ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt
     6601 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     6661 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     6721 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     6781 acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct
     6841 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     6901 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     6961 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     7021 cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac
     7081 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcact
//
