LOCUS       sequence-44437-e        8606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8606)
  AUTHORS   Loque D, Lalonde S, Looger LL, von Wiren N, Frommer WB
  TITLE     A cytosolic trans-activation domain essential for ammonium uptake.
  JOURNAL   Nature. 2007 Mar 8;446(7132):195-8. Epub 2007 Feb 11.
  PUBMED    17293878
REFERENCE   2  (bases 1 to 8606)
  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: "Nature."; 
            date: "2007-03-8"; volume: "446(7132)"; pages: "195-8. Epub 2007 Feb
            11"
COMMENT     Sequence Label: pDRf1-GW
FEATURES             Location/Qualifiers
     source          1..8606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    22..146
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(183..188,189..206,207..213)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(333..352)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1271..1576
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VXRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1455..1474
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1617..1741)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1803..1822)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1804..1822)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      1970..2157
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2025..2049)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2169..2186)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2169..2185)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2178..2200)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2621..3967
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4078..4881)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4882..5097)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5122..5140)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5208..5312
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5313..5381,5382..6173)
                     /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(5531..5550)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6344..6932
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6833..6852
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7086..7103
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7220..7241
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7256..7261,7262..7279,7280..7286)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7294..7310
                     /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     7299..7321
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7318..7334
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7318..7334
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7364..7819)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7501..7522)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7713..7732
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 actagtgaat tcgcggccat cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat
       61 gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt
      121 aaaacacaac atatccagtc actatggcgg cccctcgagg ggccgcatta ggcaccccag
      181 gctttacact ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatccgtcga
      241 gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg
      301 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta
      361 cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata
      421 agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg
      481 aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt
      541 acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg
      601 atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg
      661 cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga
      721 gtttcaccag tttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc
      781 accatgggca aatattatac gcaagggcga caaggtgctg atgccgctgg cgattcaggt
      841 tcatcatgcc gtttgtgatg gcttccatgt cgggcagaat gcttaatgaa ttacaacagt
      901 actgcgatga gtggcagggc ggggcgtaan cgcgtggatc cggcttacta aaagccagat
      961 aacagtatgc gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata
     1021 cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt attacagnga cagttgacag
     1081 cgacagctat cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca
     1141 accatgcaga atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa
     1201 gggatggctg aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg
     1261 ggctggtgaa atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt
     1321 tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc
     1381 cagtgcacgt ctgctgtcag ataaagtcnc ccgtgaactt tacccggtgg tgcatatcgg
     1441 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg
     1501 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat
     1561 gttctgggga atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata
     1621 gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat
     1681 ttaatatatt gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg
     1741 tgtagttaat tcatgatggc cgctgcaggt cgacctcgag ggggggcccg gtacccaatt
     1801 cgccctatag tgagtcgtat tacgcgcgga tccagctttg gacttcttcg ccagaggttt
     1861 ggtcaagtct ccaatcaagg ttgtcggctt gtctaccttg ccagaaattt acgaaaagat
     1921 ggaaaagggt caaatcgttg gtagatacgt tgttgacact tctaaataag cgaatttctt
     1981 atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat acaaatttta
     2041 aagtgactct taggttttaa aacganaatt cttattcttg agtaactctt tcctgtaggt
     2101 caggttgctt tctcaggtat agcatgaggt cgctcttatt gaccacacct ctaccggcat
     2161 gccaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     2221 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     2281 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat
     2341 tttctcctta cgcatctgtg cggtatttca caccgcataa tcggatcgta cttgttaccc
     2401 atcattgaat tttgaacatc cgaacctggg agttttccct gaaacagata gtatatttga
     2461 acctgtataa taatatatag tctagcgctt tacggaagac aatgtatgta tttcggttcc
     2521 tggagaaact attgcatcta ttgcataggt aatcttgcac gtcgcatccc cggttcattt
     2581 tctgcgtttc catcttgcac ttcaatagca tatctttgtt aacgaagcat ctgtgcttca
     2641 ttttgtagaa caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg
     2701 catttttaca gaacagaaat gcaacgcgaa agcgctattt taccaacgaa gaatctgtgc
     2761 ttcatttttg taaaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg
     2821 agctgcattt ttacagaaca gaaatgcaac gcgagagcgc tattttacca acaaagaatc
     2881 tatacttctt ttttgttcta caaaaatgca tcccgagagc gctatttttc taacaaagca
     2941 tcttagatta ctttttttct cctttgtgcg ctctataatg cagtctcttg ataacttttt
     3001 gcactgtagg tccgttaagg ttagaagaag gctactttgg tgtctatttt ctcttccata
     3061 aaaaaagcct gactccactt cccgcgttta ctgattacta gcgaagctgc gggtgcattt
     3121 tttcaagata aaggcatccc cgattatatt ctataccgat gtggattgcg catactttgt
     3181 gaacagaaag tgatagcgtt gatgattctt cattggtcag aaaattatga acggtttctt
     3241 ctattttgtc tctatatact acgtatagga aatgtttaca ttttcgtatt gttttcgatt
     3301 cactctatga atagttctta ctacaatttt tttgtctaaa gagtaatact agagataaac
     3361 ataaaaaatg tagaggtcga gtttagatgc aagttcaagg agcgaaaggt ggatgggtag
     3421 gttatatagg gatatagcac agagatatat agcaaagaga tacttttgag caatgtttgt
     3481 ggaagcggta ttcgcaatat tttagtagct cgttacagtc cggtgcgttt ttggtttttt
     3541 gaaagtgcgt cttcagagcg cttttggttt tcaaaagcgc tctgaagttc ctatactttc
     3601 tagctagaga ataggaactt cggaatagga acttcaaagc gtttccgaaa acgagcgctt
     3661 ccgaaaatgc aacgcgagct gcgcacatac agctcactgt tcacgtcgca cctatatctg
     3721 cgtgttgcct gtatatatat atacatgaga agaacggcat agtgcgtgtt tatgcttaaa
     3781 tgcgtactta tatgcgtcta tttatgtagg atgaaaggta gtctagtacc tcctgtgata
     3841 ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc actacccttt agctgttcta
     3901 tatgctgcca ctcctcaatt ggattagtct catccttcaa tgctatcatt tcctttgata
     3961 ttggatcgat ccgatgataa gctgtcaaac atgagaattg ggtaataact gatataatta
     4021 aattgaagct ctaatttgtg agtttagtat acatgcattt acttataata cagtttttta
     4081 gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc
     4141 ctctacctta gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga
     4201 tcctgtagag accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc
     4261 atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat
     4321 gtctctttga gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt
     4381 acccttagta tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa
     4441 aaggcctcta ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc
     4501 tgggcccacc acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc
     4561 cgcagagtac tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag
     4621 taaaaaattg tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa
     4681 atcagtcaag atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac
     4741 taactccagt aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt
     4801 ttcgtgcatg atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc
     4861 cttatatgta gctttcgaca tgatttatct tcgtttcctg catgtttttg ttctgtgcag
     4921 ttgggttaag aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac
     4981 caatctaagt ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa
     5041 aaaatttcaa ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaaaag
     5101 ctaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     5161 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5221 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5281 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5341 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5401 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5461 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5521 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5581 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5641 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5701 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5761 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5821 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5881 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5941 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6001 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6061 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6121 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6181 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6241 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6301 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6361 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6421 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6481 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6541 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6601 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6661 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6721 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6781 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6841 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6901 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6961 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7021 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     7081 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     7141 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     7201 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     7261 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     7321 gaaacagcta tgaccatgat tacgccaagc ttaccgcatc aggaaattgt aagcgttaat
     7381 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     7441 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     7501 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     7561 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     7621 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     7681 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     7741 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     7801 gcgccgctac agggcgcgtc cattcgccaa gcttcctgaa acggagaaac ataaacaggc
     7861 attgctggga tcacccatac atcactctgt tttgcctgac cttttccggt aatttgaaaa
     7921 caaacccggt ctcgaagcgg agatccggcg ataattaccg cagaaataaa cccatacacg
     7981 agacgtagaa ccagccgcac atggccggag aaactcctgc gagaatttcg taaactcgcg
     8041 cgcattgcat ctgtatttcc taatgcggca cttccaggcc tcgatcgaga ccgtttatcc
     8101 attgcttttt tgttgtcttt ttccctcgtt cacagaaagt ctgaagaagc tatagtagaa
     8161 ctatgagctt tttttgtttc tgttttcctt tttttttttt ttacctctgt ggaaattgtt
     8221 actctcacac tctttagttc gtttgtttgt tttgtttatt ccaattatga ccggtgacga
     8281 aacgtggtcg atggtgggta ccgcttatgc tcccctccat tagtttcgat tatataaaaa
     8341 ggccaaatat tgtattattt tcaaatgtcc tatcattatc gtctaacatc taatttctct
     8401 taaatttttt ctctttcttt cctataacac caatagtgaa aatctttttt tcttctatat
     8461 ctacaaaaac tttttttttc tatcaacctc gttgataaat tttttcttta acaatcgtta
     8521 ataattaatt aattggaaaa taaccatttt ttctctcttt tatacacaca ttcaaaagaa
     8581 agaaaaaaaa tataccccag cctcga
//
