LOCUS       sequence-482406-        5715 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Over-express FLAG-rad24 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5715)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 5715)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Sc FLAG-rad24-pRSFDuet
FEATURES             Location/Qualifiers
     source          1..5715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      323..1072
                     /direction=RIGHT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     803..822
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1045..1062
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1088..1179
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1180..1995
                     /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             1180..1995
                     /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     complement(1253..1272)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(2225..2272)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2268..2286
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2324..2368)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(4481..4546)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    4593..4617
                     /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(4617..4636)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4618..4636)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        4682..4759
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4760..5715,1..127)
                     /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     complement(4779..4798)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
       61 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
      121 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac
      181 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
      241 acatgagaat tacaacttat atcgtatggg gctgacttca ggtgctacat ttgaagagat
      301 aaattgcact gaaatctaga gtaacggaat agctgttcgt tgacttgata gaccgattga
      361 ttcatcatct cataaataaa gaaaaaccac cgctaccaac ggtggttttc tcaaggttcg
      421 ctgagctacc aactctttga accaaggtaa gtgggttgga ggaccgcact caccaaaatc
      481 tgttctttca gtttagcctt aacaggtgca taacttcaag acaaagtcct ctaaatcagt
      541 taccaatggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      601 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      661 tggagcgaac gacctacacc gaactgagat accaacagcg tgagctatga gaaagcgcca
      721 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      781 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      841 gccacctctg gcttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      901 aaaacgcctg cggcgttggc ttcttccggt gctttgcttt ttgctcacat gttctttccg
      961 gctttatccc ctgattctgt ggataaccgt attaccgctt ttgagtgagc tgacaccgct
     1021 cgccgcagtc gaacgaccga gcgtagcgag tcagtgagcg aggaagcgga agagcgctgc
     1081 atgcctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1141 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagccatat tcaacgggaa
     1201 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     1261 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     1321 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     1381 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     1441 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     1501 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     1561 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga ccgcgtattt
     1621 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     1681 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     1741 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     1801 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     1861 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     1921 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     1981 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     2041 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacttgcgg agacccggtc
     2101 gtcagcttgt cgtcggttca gggcagggtc gttaaatagc cgcttatgtc tattgctggt
     2161 ttaccggttt attgactacc ggaagcagtg tgaccgtgtg cttctcaaat gcctgaggtt
     2221 tcagcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct agttattgct
     2281 cagcggtggc agcagcctag gttaattaag ctgcgctagt agacgagtcc atgtgctggc
     2341 gttcaaattt cgcagcagcg gtttctttac cagactcgag ggtaccgacg tcagcgatcg
     2401 cgtggccggc cgatatccaa ttgagatcat agatttgtgt ggaatatttc ctggggtttt
     2461 ctcgtcaaat ttaaagagta aaaagttaga gtatttccag atctgaatct gaaagggact
     2521 cactgataac tggcgcttta cgcggctgtc tctgggacag aatttcacat agagactcgt
     2581 cttcgtcgct tctaccaaat gtttcattgc caatattatt gctgttatcg ctatcgctat
     2641 cgacaatagg gtcatcatta aaagaagttt cttcgtcttc aagatcgtcg ttagccatca
     2701 tcacatttct ttcatattgg tcaatcaaca tgcgaagcct tctgtccctt tccttttttt
     2761 gatcctctaa atggtcacaa ttattgctat cattatccat caatggtgct agtccatcct
     2821 ccacagatag tgcttcgata ggcccgccta tccgatccac aacgtcgatt ccgttctcaa
     2881 ctttcattat atcactaaat ttgtccatgg tttgtttggg ccccgaggag ccactcggaa
     2941 gattcttcaa ataatagaga atgtattttt ttttataact ctgacacttt ctaattagag
     3001 gtgcgtagta gccaaattct agagttatat tcctgaaaga atgtaccgcg ttgtacttat
     3061 aaagactaac atttaaccaa tcttcagctt gaactttaaa tgaattttgt aattttctta
     3121 ctttccattc tcttggaaaa taaaccgttc catgattatg gccttgttta ctgatgttac
     3181 gaaaggtttt gcgcacttct cgtaaaccat actcattgga ttctggtaga ccattcatat
     3241 tatcacactc gctcaggcaa tccacaattg atgatgcatc agaaatgctg aattcgcctt
     3301 tattaaatgt gttatagttc tctaatattc ctaatttgaa atcctctttc gataacaaat
     3361 tgttcgaatt ttcgaagagg ttattaatca tttcgttatc gttattcgtg ctatgggaac
     3421 catgtatcac cttcccaatg gcatgaaagt atgatatggt ggattctcgg gttgaaatcg
     3481 gcaaacttcc acttgatgtc gcccaaaatt gaagggtcgt aatggccgac ctaatatcac
     3541 cagtctcttg cgcaatataa tctatgactt cctgtctttt attccattta tttttctcct
     3601 tcaacatttt catattctgt acacaaataa atttcaagtg cttttttaat aaagtgctgt
     3661 taattggatt aaacttaatt cttttcaacc ttggatgcat caatatttct ttgttcatta
     3721 tggtttctgc actaaatgta taatcaatac caaatttgcg ataattatta tcgttctctg
     3781 gaatttcaca ttcagttata catataacaa ggggaggtaa tagaggctcc gaactatata
     3841 gccactgtaa tataagttgt tgaaatcgac gtctggtatc tatatggaag acgttgggaa
     3901 ggtcctcgat aagtattaat gacaggttgg acatcacaag ataccgtgcg ccttttaaga
     3961 actcactaaa gctttccatc tgaggaagat cgttgactat acaatcacct ctaaactcgg
     4021 tcactttatg ctcgttcggg gtgcttcgaa aggacgttcc gttgctgttt tgtctgtatt
     4081 taggaactaa gatttttgag agttctttta tgaccgtact tttagagcat ccactggggc
     4141 cagacagtag taggatccta tgcttggcgt taggtaaaaa catggcatct aaagcttctt
     4201 gtacatcttt aagttttctt ttatgtatgg ccacttgctc caaacaattg ggcttgaact
     4261 tttcgtacca ctgttcaccg ccgtcgtcgt tgatacttgg gaggatacta gacgtatctt
     4321 gcttggaaag aaagtcattt tcagtgcttc tcgccttacg aactggcgaa gtcggtctag
     4381 atgagctcca ttttgttatt tgcgagccca atgaactgag actatattgt aataagggcc
     4441 gtttgttcaa attcgtacta tccatatgac tagtggatcg cttgtcatcg tcatccttgt
     4501 agtcgatgtc atgatcttta taatcaccgt catggtcttt gtagtccatg gtatatctcc
     4561 ttattaaagt taaacaaaat tatttctaca ggggaattgt tatccgctca caattcccct
     4621 atagtgagtc gtattaattt cctaatgcag gagtcgcata agggagagcg tcgagatccc
     4681 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     4741 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     4801 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     4861 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     4921 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     4981 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     5041 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     5101 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     5161 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     5221 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     5281 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     5341 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     5401 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     5461 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     5521 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggac atctcggtag tgggatacga
     5581 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     5641 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     5701 gggcaatcag ctgtt
//
