LOCUS       sequence-472706-        6709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  over-expresses C-terminal FLAG tagged E. coli delta (Y316A) in E. 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6709)
  TITLE     O'Donnell Lab Batch 2
REFERENCE   2  (bases 1 to 6709)
  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"
COMMENT     Sequence Label: Ec holA(Y316A)-FLAG-pET(11a)(ACH-)
FEATURES             Location/Qualifiers
     source          1..6709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..33
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    48..187
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(113..132)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     273..295
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(289..480)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1792..2874)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2836..2855
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2875..2952)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3158..3177
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3261..3280
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3261..3279
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3280..3304
                     /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)."
     CDS             4355..4420
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4474..4492)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4488..4535
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4765..4783)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4847..4951
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4952..5020,5021..5812)
                     /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(5170..5189)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5983..6571
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6472..6491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
       61 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
      121 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
      181 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      241 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
      301 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
      361 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
      421 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
      481 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
      541 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
      601 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
      661 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
      721 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
      781 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
      841 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
      901 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
      961 gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg
     1021 ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg
     1081 agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc
     1141 catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat
     1201 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
     1261 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
     1321 tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg
     1381 agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag
     1441 cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     1501 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     1561 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     1621 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     1681 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     1741 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     1801 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1861 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     1921 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     1981 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     2041 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     2101 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     2161 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     2221 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     2281 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     2341 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     2401 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     2461 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     2521 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     2581 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     2641 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     2701 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     2761 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     2821 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     2881 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     2941 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     3001 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     3061 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     3121 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     3181 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     3241 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     3301 ttcccctcta gaaggagata taaatatgat tcggttgtac ccggaacaac tccgcgcgca
     3361 gctcaatgaa gggctgcgcg cggcgtatct tttacttggt aacgatcctc tgttattgca
     3421 ggaaagccag gacgctgttc gtcaggtagc tgcggcacaa ggattcgaag aacaccacac
     3481 tttttccatt gatcccaaca ctgactggaa tgcgatcttt tcgttatgcc aggctatgag
     3541 tctgtttgcc agtcgacaaa cgctattgct gttgttacca gaaaacggac cgaatgcggc
     3601 gatcaatgag caacttctca cactcaccgg acttctgcat gacgacctgc tgttgatcgt
     3661 ccgcggtaat aaattaagca aagcgcaaga aaatgccgcc tggtttactg cgcttgcgaa
     3721 tcgcagcgtg caggtgacct gtcagacacc ggagcaggct cagcttcccc gctgggttgc
     3781 tgcgcgcgca aaacagctca acttagaact ggatgacgcg gcaaatcagg tgctctgcta
     3841 ctgttatgaa ggtaacctgc tggcgctggc tcaggcactg gagcgtttat cgctgctctg
     3901 gccagacggc aaattgacat taccgcgcgt tgaacaggcg gtgaatgatg ccgcgcattt
     3961 cacccctttt cattgggttg atgctttgtt gatgggaaaa agtaagcgcg cattgcatat
     4021 tcttcagcaa ctgcgtctgg aaggcagcga accggttatt ttgttgcgca cattacaacg
     4081 tgaactgttg ttactggtta acctgaaacg ccagtctgcc catacgccac tgcgtgcgtt
     4141 gtttgataag catcgggtat ggcagaaccg ccggggcatg atgggcgagg cgttaaatcg
     4201 cttaagtcag acgcagttac gtcaggccgt gcaactcctg acacgaacgg aactcaccct
     4261 caaacaagat gccggtcagt cagtgtgggc agagctcgag gggttatctc ttctgttgtg
     4321 ccataaaccc ctggcggacg tatttatcga cggtgactac aaagaccatg acggtgatta
     4381 taaagatcat gacatcgact acaaggatga cgatgacaag tagggatccg gctgctaaca
     4441 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     4501 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     4561 atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt
     4621 gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg
     4681 ttagcaattt aactgtgata aactaccgca ttaaagctgg tcgatgataa gctgtcaaac
     4741 atgagaattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     4801 tgataataat ggtttcttag caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4861 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4921 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4981 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5041 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5101 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5161 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     5221 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5281 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5341 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5401 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5461 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     5521 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5581 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5641 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5701 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5761 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5821 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5881 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5941 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6001 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6061 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6121 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6181 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6241 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6301 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6361 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6421 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6481 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6541 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6601 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6661 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagc
//
