LOCUS       sequence-242453-        7569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for genomic integration of CggR_R250A repressor regulated by
            the pTEFmut7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7569)
  AUTHORS   Monteiro F, Hubmann G, Takhaveev V, Vedelaar SR, Norder J, Hekelaar 
            J, Saldida J, Litsios A, Wijma HJ, Schmidt A, Heinemann M
  TITLE     Measuring glycolytic flux in single yeast cells with an orthogonal 
            synthetic biosensor.
  JOURNAL   Mol Syst Biol. 2019 Dec;15(12):e9071. doi: 10.15252/msb.20199071.
  PUBMED    31885198
REFERENCE   2  (bases 1 to 7569)
  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"; journalName: "Mol Syst 
            Biol."; date: "2019-12"; volume: "15(12):e9071. doi"; pages: " 
            10.15252/msb.20199071"
COMMENT     Sequence Label: pHO_pTEFmut7_CggR_R250A_ble
FEATURES             Location/Qualifiers
     source          1..7569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1436..1468
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(1472..1480,1481..1495)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(2560..2576)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2800..2833
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2888..3231
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      3624..3821
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    3884..3917
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     4502..4518
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4502..4518
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4532..4551)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4533..4551)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4558..4574)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4874..4893)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5006..5027
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5181..5200)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5300..5322
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     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
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     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"
     primer_bind     7324..7341
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7458..7479
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    7534..7550
                     /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     7539..7561
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(7558..7569,1..5)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(7558..7569,1..5)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgaccatga ttacgccact agtccgaggc ctcgagatcc gatatcgccg tggcggccgc
       61 cagctgaagc ttaattatcc tgggcacgag tgaaacaaag ctaaaacctt tatttagcat
      121 ggccattgaa tgtaacaatt atatatatcg caagcacaaa aaatcaagga gagagaacta
      181 ccactttgtt catgtgtaca atgttcatta tctccataag caaaaaaaaa aaaatagaaa
      241 acatatgcta taaggttgat attctcacga gtaagcggca cttgctactt attgacattg
      301 cagatttttg gctacagaaa tagtatatta gagattataa ttgctaatca aatcaaaata
      361 taaaattagt aaaccaaacc atttataccc ttccttagta gttatggatt gttttttaat
      421 gatatttctg caaaccaaag aaagattgtt atccagatag aatttagttt tgatattcat
      481 ttttttgttg aagattgaac gccatatctg ggcctcataa ttcaaaagac ggtgccatta
      541 tcggtagcgt ttcgcattgt actggatttc agaaatttca cagttgatga atcgaaaaga
      601 atggtctcat tgcaacacgt aaggttaaga tgtccctttt taccattata ggcaataaat
      661 gaatcataaa acgaccgtat actggtgaaa tagtagggag aacgagtacc tgtagtaaaa
      721 agtataaatc atagttaatc gggcaatgtc cctcgatcaa ggagtattgt gtcatgttcg
      781 agacaaacgc caacattttt gtttcttttg gacaaatgtt gtttgcattt atgatccgtt
      841 atattttgat ctaatgtaga gttgcacgta gttcttactg gcaaagaaat cgatgcatac
      901 caaaaaagaa taaaggtgat atttgatctt taccgtttag ttccaacgta aaattgtgcc
      961 tttggactta aaatggcgtc gtacgctagg tcgagatagc ttcaaaatgt ctctactcct
     1021 tttttactct tccagatttt ctcggactcc gcgcaccgcc gtaccacttc aaaacaccca
     1081 agcacagcat actaaattcc ccctcctcct tcctctaggg tgccgttaat tacccgtact
     1141 aaaggtttgg aaaaggaaaa agagaccgcc tcgtcccttt ttcttcgtcg gagaaggcaa
     1201 taaaaatttt tatcacgttt ctttctcttg aaaacttttt tttcgatttt gttctctttc
     1261 gacgacctcc cattgatatt tgagttaaca aacggtcttc aatttctcaa gtttcagctt
     1321 catttttcct gttctattac aacttttttt acttcttgct cattggaaag aaagcatagc
     1381 aatctaatct aagttttcta gaaaaatgcg gggttctcat catcatcatc atggtatggc
     1441 tagcatgact ggtggacagc aaatgggtcg ggatctgtac gacgatgacg ataaggatca
     1501 tcccttcacc atgaatcaat tgatccaagc ccaaaagaag ttgttgccag atttgttgtt
     1561 ggtcatgcaa aagagattcg aaatcttgca atatatcaga ttgaccgaac ctatcggtag
     1621 aagatctttg tctgcttctt tgggtatctc cgaaagagtt ttgagaggtg aagtccaatt
     1681 cttgaaagaa caaaacttgg tcgacatcaa gactaacggt atgactttga ctgaagaagg
     1741 ttacgaatta ttgtccgtct tggaagatac catgaaggat gttttgggtt tgaccttgtt
     1801 ggaaaagact ttgaaagaaa gattgaactt gaaggacgcc atcatcgttt ctggtgattc
     1861 tgatcaatct ccatgggtca aaaaagaaat gggtagagct gctgttgctt gcatgaagaa
     1921 aagattttct ggtaagaaca tcgttgctgt tactggtggt actactattg aagctgttgc
     1981 tgaaatgatg accccagatt ctaagaacag agaattgttg tttgttccag ctagaggtgg
     2041 tttgggtgaa gatgttaaga atcaagctaa caccatttgc gctcatatgg ctgaaaaagc
     2101 ttcaggtact tacagattgt tattcgtccc aggtcaattg tctcaaggtg cttactcttc
     2161 cattatcgaa gaaccatctg tcaaagaagt cttgaacacc atcaaatccg cttccatgtt
     2221 ggttcacggt attggtgaag ctaaaactat ggctcaagca agaaacaccc cattggaaga
     2281 tttgaaaaag atcgatgata acgatgctgt taccgaagct ttcggttact actttaatgc
     2341 tgatggtgaa gttgttcaca aggttcattc agttggtatg caattggatg atattgatgc
     2401 catcccagat attattgcag ttgctggtgg ttcttctaag gctgaagcaa ttgaagctta
     2461 cttcaagaag ccaagaaaca ctgttttggt tactgatgaa ggtgctgcca aaaagttgtt
     2521 gagagatgaa tgatagctcg aggtaccgag ctcgaattca ctggccgtcg ttttacaacg
     2581 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     2641 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2701 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtacgcc
     2761 agctgaagct tcgtacgctg caggtcgaca acccttaata taacttcgta taatgtatgc
     2821 tatacgaagt tattaggtct agagatctgt ttagcttgcc tcgtccccgc cgggtcaccc
     2881 ggccagcgac atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg
     2941 ggcatgatgt gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc
     3001 atccatacat tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac
     3061 ctgcgagcag ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct
     3121 gtagagaaat ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt
     3181 taaaatcttg ctaggataca gttctcacat cacatccgaa cataaacaac catgggtatg
     3241 accgaccaag cgacgcccaa cctgccatca cgagatttcg atcccaccgc cgccttctat
     3301 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     3361 gatctcatgc tggagttctt cgcccacccc gggctcgatc ccctcgcgag ttggttcagc
     3421 tgctgcctga ggctggacga cctcgcggag ttctaccggc agtgcaaatc cgtcggcatc
     3481 caggaaacca gcagcggcta tccgcgcatc catgcccccg aactgcagga gtggggaggc
     3541 acgatggccg ctttggtcga cccggacggg acgctcctgc gcctgataca gaacgaattg
     3601 cttgcaggca tctcatgatc agtactgaca ataaaaagat tcttgttttc aagaacttgt
     3661 catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt
     3721 atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa
     3781 tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac
     3841 gccgccatcc agtgtcgaaa acgagctctc gagaaccctt aatataactt cgtataatgt
     3901 atgctatacg aagttattag gtgatatcag atccactagt ggcctatgcg cgccgccatc
     3961 cagtgtcgaa aacgagctcg aattcctggg ggaacaactt cacagaatgt tttgtcatat
     4021 tgtcgaagtg gtcacaaaac aagagaagtt ccgccaatta taaaaaggga acccgtatat
     4081 ttcagcttca cggatgattt ccagggtgag agtactgtat atgggcttac gatagaaggc
     4141 cataaaaatt tcttgcttgg caacaaaata gaagtgaaat catgtcgagg ctgctgtgtg
     4201 ggagaacagc ataaaatatc acaaaaaaag aatctaaaac actgtgttgc ttgtcccaga
     4261 aagggaatca agtattttta taaagattgg agtggtaaaa atcgagtatg tgctagatgc
     4321 tatggaagat acaaattcag cggtcatcac tgtataaatt gcaagtatgt accagaagca
     4381 cgtgaagtga aaaaggcaaa agacaaaggc gaaaaattgg gcattacgcc cgaaggtttg
     4441 ccagttaaag gaccagagtg tataaaatgt ggcggaatct tacagtggcc tatgcggccg
     4501 ctctagaact agtggatcga tccccaattc gccctatagt gagtcgtatt acaattcact
     4561 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4621 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4681 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     4741 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     4801 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4861 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4921 ccccgtcaag ctctaaatcg gggtgggcca tcgccctgat agacggtttt tcgccctttg
     4981 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5041 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     5101 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     5161 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     5221 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5281 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5341 aaacgcgcga 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 cgtattgacg 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 cctgtagcaa 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 aatactgttc 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 acgcggcctt tttacggttc
     7201 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     7261 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     7321 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     7381 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     7441 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     7501 tttatgcttc cgcggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     7561 gaaacagct
//
