LOCUS       sequence-482407-        5188 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Over-express Sc FLAG-PKA kinase motif-mec3 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5188)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 5188)
  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-kin-mec3-pRSFDuet
FEATURES             Location/Qualifiers
     source          1..5188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      883..1632
                     /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     1363..1382
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1605..1622
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1648..1739
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1740..2555
                     /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             1740..2555
                     /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(1813..1832)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(2785..2832)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2828..2846
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2884..2928)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(4454..4465)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(4514..4579)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    4626..4650
                     /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(4650..4669)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4651..4669)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        4715..4792
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4793..5188,1..687)
                     /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(4812..4831)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
       61 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
      121 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
      181 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
      241 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
      301 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
      361 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggacatctc ggtagtggga
      421 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
      481 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
      541 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
      601 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      661 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
      721 ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg
      781 gtgggcgcgg ggcatgacta acatgagaat tacaacttat atcgtatggg gctgacttca
      841 ggtgctacat ttgaagagat aaattgcact gaaatctaga gtaacggaat agctgttcgt
      901 tgacttgata gaccgattga ttcatcatct cataaataaa gaaaaaccac cgctaccaac
      961 ggtggttttc tcaaggttcg ctgagctacc aactctttga accaaggtaa gtgggttgga
     1021 ggaccgcact caccaaaatc tgttctttca gtttagcctt aacaggtgca taacttcaag
     1081 acaaagtcct ctaaatcagt taccaatggc tgctgccagt ggcgataagt cgtgtcttac
     1141 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     1201 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat accaacagcg
     1261 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1321 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     1381 ttatagtcct gtcgggtttc gccacctctg gcttgagcgt cgatttttgt gatgctcgtc
     1441 aggggggcgg agcctatgga aaaacgcctg cggcgttggc ttcttccggt gctttgcttt
     1501 ttgctcacat gttctttccg gctttatccc ctgattctgt ggataaccgt attaccgctt
     1561 ttgagtgagc tgacaccgct cgccgcagtc gaacgaccga gcgtagcgag tcagtgagcg
     1621 aggaagcgga agagcgctgc atgcctattt gtttattttt ctaaatacat tcaaatatgt
     1681 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1741 tgagccatat tcaacgggaa acgtcttgct ctaggccgcg attaaattcc aacatggatg
     1801 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     1861 atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     1921 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc
     1981 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     2041 tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg
     2101 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt
     2161 ttaacagcga ccgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg
     2221 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     2281 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     2341 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     2401 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     2461 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     2521 tgcagtttca tttgatgctc gatgagtttt tctaagaatt aattcatgag cggatacata
     2581 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     2641 ccacttgcgg agacccggtc gtcagcttgt cgtcggttca gggcagggtc gttaaatagc
     2701 cgcttatgtc tattgctggt ttaccggttt attgactacc ggaagcagtg tgaccgtgtg
     2761 cttctcaaat gcctgaggtt tcagcaaaaa acccctcaag acccgtttag aggccccaag
     2821 gggttatgct agttattgct cagcggtggc agcagcctag gttaattaag ctgcgctagt
     2881 agacgagtcc atgtgctggc gttcaaattt cgcagcagcg gtttctttac cagactcgag
     2941 ggtaccgacg tcagcgatcg cgtggccggc cgatatccaa ttgagatcct tagcaacgta
     3001 gcaaagaaat gtaccgctgt agggtttaca agcccttcga tcttgctata taatatatga
     3061 tttgtcctct ttcccttggt ttttccacat cttcgctatc ttccaagcta ccacgatcca
     3121 acgaacagtg gaatacgcag gattcgtcgt gagaaatcgc caaaacaact tcttcaaatg
     3181 cagcgtataa cttggaacac accttccaat ctttgcaacg gatgattact tcatgtgtgg
     3241 acgaactttc ctgttcagcc ttttccacca taacggatat gtcattaaat tcagtatcac
     3301 cgctagtatc agctgtgtaa atgtttcccc gcgtatctgc gatcgagcta tcctcaattc
     3361 ttaataaatc ttcatcgtag cggatatttt cttccatctc tcgatctcta gtattggtat
     3421 atagtgaaga catcggttta tccgcttcga taatcggaag agatccttcc tcctgccggc
     3481 cgtctgtgtc gatgtgctgg ttttgggaag gattgtcagt gagcccttct tggcgttgta
     3541 tcacagagtc taagggtcca ttccagcata tttccaaatg ccaatctaat tcattcacaa
     3601 ttatcttaag ttctacatca tcaccttcat ttccatgctc cttttttttg actcccatta
     3661 aatgaatgtg gttgacatta ctgtaccgtt caacacgtct aataaacccg tggaaggcgg
     3721 agccaaactc gcccgatatt ggtggcagct tgtacatcat cagttgaata tagttaatca
     3781 ttggctcttg tattcgcgta tcttgtgctc ggaataatag tttgacaggt actttgaacg
     3841 aatgcatgat aaccttattg cttgcgagta gatttcctgt gcctactgtg gttggtaaac
     3901 cattgtgctc atccactccc ccattcataa ctatagcgtc atttgggccg ctagtatgta
     3961 caatttcttc gaatgttata cctagagcac aaatggggtt cggttttgag gttgtatcta
     4021 cgcctcctgc tgttcctcct gaaagtgttc cgttatttgt attccattct ggcattgact
     4081 gtagttttat agtcatatta gaggaagatc cttggttcat tactcgatca taccttttaa
     4141 acacactcaa taaagaatca caattacact ccattgttat tgtattgagc tcgcgagctg
     4201 atataactgt atataatctg aatacatcat gaggaatggt acaccaaagc tgaccagtat
     4261 cccctcgtaa tattgtaccg ttgttactgc tgttgagtga tgattttgga gtggatatta
     4321 ttgtcaatct ttcactatta aatcttaaga tagccgtctt tcgtagcgaa gcaactgtat
     4381 tgatagtagt tcttagcaat ttataatcat caggtgcttc acaaccattt actatcaatt
     4441 ttaatttcat atggcggcct tcaatatggc cgctgctcac gctcgcgcgg cgcagaatat
     4501 gactagtgga tcgcttgtca tcgtcatcct tgtagtcgat gtcatgatct ttataatcac
     4561 cgtcatggtc tttgtagtcc atggtatatc tccttattaa agttaaacaa aattatttct
     4621 acaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcctaatg
     4681 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
     4741 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     4801 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     4861 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     4921 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     4981 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     5041 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     5101 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     5161 cattaactat ccgctggatg accaggat
//
