LOCUS       sequence-482111-        7181 bp    DNA     circular SYN 06-AUG-2025
DEFINITION  Over-express Ec FLAG-S6-dnaB subunit in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7181)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 7181)
  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-S6-dnaB-pET
FEATURES             Location/Qualifiers
     source          1..7181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     143..160
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    175..314
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(240..259)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     400..422
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(416..607)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2962..2981
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3001..3078)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3284..3303
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3387..3406
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3387..3405
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3406..3430
                     /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)."
     RBS             3445..3467
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3477..3542
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(5069..5087)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5083..5130
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5305..5310,5311..5327,5328..5333))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5360..5378)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5446..5550
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5551..5619,5620..6411)
                     /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(5769..5788)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6582..7170
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7071..7090
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
       61 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      121 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      181 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      241 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      301 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      361 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
      421 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
      481 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
      541 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
      601 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
      661 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
      721 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
      781 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
      841 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
      901 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
      961 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1021 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     1081 acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata
     1141 tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa
     1201 ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc
     1261 ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac
     1321 aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag
     1381 cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg
     1441 atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc
     1501 ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa
     1561 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     1621 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     1681 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     1741 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     1801 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     1861 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     1921 actgcccgct ttccagtcgg gaaactgtcg tgccagctgc attaatgaat cggccaacgc
     1981 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     2041 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     2101 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     2161 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     2221 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     2281 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     2341 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     2401 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     2461 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     2521 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     2581 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     2641 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     2701 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     2761 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     2821 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     2881 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     2941 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     3001 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     3061 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     3121 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     3181 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     3241 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     3301 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     3361 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     3421 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatggact
     3481 acaaagacca tgacggtgat tataaagatc atgacatcga ctacaaggat gacgatgaca
     3541 agcgatccac tagtcatatc ggcgatagcc tgagctggct gctgcgcctg ctgaaccata
     3601 tggcaggaaa taaacccttc aacaaacagc aggctgaacc ccgcgaacgc gatccacaag
     3661 ttgccgggct gaaagtgcct ccgcactcga tcgaagcgga gcagtcggtg ttgggcggtt
     3721 taatgctaga taacgaacgc tgggatgatg tagccgagcg tgtggtagca gacgattttt
     3781 acacccgccc acaccgtcat atctttactg aaatggcgcg tttgcaggaa agcggtagcc
     3841 ctatcgatct gattactctt gcggaatcgc tggaacgcca ggggcaactc gatagcgtcg
     3901 gtggttttgc ttatctggca gagctgtcaa aaaatacgcc aagtgcggct aacatcagtg
     3961 cctatgcgga catcgtgcgt gaacgtgccg ttgtccgtga gatgatctcg gttgcgaatg
     4021 agattgccga agctggtttt gatccgcagg ggcgtaccag cgaagatctg ctggatctgg
     4081 ctgaatcccg cgtctttaaa attgccgaaa gtcgtgcgaa caaagacgaa gggccgaaga
     4141 acatcgccga tgtgctcgac gcaaccgtgg cgcgtattga gcagttgttt cagcagccac
     4201 acgatggcgt taccggggta aacaccggtt atgacgatct caacaaaaaa accgctggct
     4261 tgcagccgtc ggatttgatc atcgtcgccg cgcgtccgtc gatgggtaaa acaacatttg
     4321 cgatgaacct cgtcgaaaac gcggcgatgt tgcaggataa accggtactt atcttctcgc
     4381 tggagatgcc atcagaacag atcatgatgc gttctctggc gtcgctgtcg cgcgttgacc
     4441 agactaaaat ccgtaccggg cagctcgatg acgaagactg ggcgcgcatt tccggcacca
     4501 tgggtatttt gctcgaaaaa cgcaatatct atatcgatga ctcctccggc ctgacgccaa
     4561 cggaagtgcg ttcccgcgca cgccgtattg cccgtgaaca cggcggcatc gggcttatca
     4621 tgatcgacta cctgcaactg atgcgcgtac cggcgctttc cgataaccgt acgctggaaa
     4681 ttgcagaaat ctctcgctcg ctgaaagcac tggcgaaaga actgaacgtg ccggtggtgg
     4741 cgctgtccca gttgaaccgt tctctggaac aacgtgccga caaacgcccg gtcaactccg
     4801 acctgcgtga atctggctct atcgagcagg atgcggactt gatcatgttt atctatcgtg
     4861 atgaggtgta tcacgaaaac agtgatttaa aaggcatcgc ggaaattatt atcggtaaac
     4921 aacgtaacgg cccaatcggg acggtacgcc tgacctttaa cggtcaatgg tcgcgcttcg
     4981 acaactatgc ggggccgcag tacgacgacg aataataagg atccggctgc taacaaagcc
     5041 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     5101 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc
     5161 gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg
     5221 tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc
     5281 aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag
     5341 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     5401 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5461 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5521 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5581 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5641 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5701 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5761 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
     5821 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5881 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5941 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6001 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6061 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     6121 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6181 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6241 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6301 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6361 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6421 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6481 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6541 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6601 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6661 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6721 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6781 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6841 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6901 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6961 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7021 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7081 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7141 tgctcgtcag gggggcggag cctatggaaa aacgccagca a
//
