LOCUS       sequence-370241-        7257 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning Vector and pDEST.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7257)
  AUTHORS   Ramos Baez R, Buckley Y, Yu H, Chen Z, Gallavotti A, Nemhauser JL, 
            Moss BL
  TITLE     A Synthetic Approach Allows Rapid Characterization of the Maize 
            Nuclear Auxin Response Circuit.
  JOURNAL   Plant Physiol. 2020 Apr;182(4):1713-1722. doi: 10.1104/pp.19.01475. 
            Epub 2020 Mar 2.
  PUBMED    32123041
REFERENCE   2  (bases 1 to 7257)
  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"; doi: 
            "10.1104/pp.19.01475"; journalName: "Plant Physiol"; date: 
            "2020-04"; volume: "182"; issue: "4"; pages: "1713-1722"
COMMENT     Sequence Label: pGP8GF-ccdB
FEATURES             Location/Qualifiers
     source          1..7257
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     454..473
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(692..796)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     864..882
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(920..942)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1042..1061
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1214..1401
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             1402..2064
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     terminator      2065..2230
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2120..2144)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        2253..2896
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     2790..2813
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     2902..2918
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2902..2918
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        join(2950..2955,2956..2973,2974..2980)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3034..3693
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3100..3119)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4035..4340
                     /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
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4219..4238
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4360..4425
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4426..4443
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(4491..4615)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4648..4664)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4649..4665)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      4667..4913
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4683..4701)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5927..5943)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5927..5943)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5940..5962)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5951..5967
                     /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)."
     promoter        complement(join(5975..5981,5982..5999,6000..6005))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6020..6041
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6158..6175)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6329..6917)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6409..6428)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7088..7257,1..622,623..691))
                     /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"
ORIGIN
        1 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
       61 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
      121 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
      181 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
      241 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
      301 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
      361 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
      421 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
      481 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
      541 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
      601 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
      661 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
      721 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
      781 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
      841 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
      901 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
      961 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     1021 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccataatt
     1081 cgtttaaacc gttttaagag cttggtgagc gctaggagtc actgccaggt atcgtttgaa
     1141 cacggcatta gtcagggaag tcataacaca gtcctttccc gcaattttct ttttctatta
     1201 ctcttggcct cctctagtac actctatatt tttttatgcc tcggtaatga ttttcatttt
     1261 tttttttcca cctagcggat gactcttttt ttttcttagc gattggcatt atcacataat
     1321 gaattataca ttatataaag taatgtgatt tcttcgaaga atatactaaa aaatgagcag
     1381 gcaagataaa cgaaggcaaa gatgacagag cagaaagccc tagtaaagcg tattacaaat
     1441 gaaaccaaga ttcagattgc gatctcttta aagggtggtc ccctagcgat agagcactcg
     1501 atcttcccag aaaaagaggc agaagcagta gcagaacagg ccacacaatc gcaagtgatt
     1561 aacgtccaca caggtatagg gtttctggac catatgatac atgctctggc caagcattcc
     1621 ggctggtcgc taatcgttga gtgcattggt gacttacaca tagacgacca tcacaccact
     1681 gaagactgcg ggattgctct cggtcaagct tttaaagagg ccctaggggc cgtgcgtgga
     1741 gtaaaaaggt ttggatcagg atttgcgcct ttggatgagg cactttccag agcggtggta
     1801 gatctttcga acaggccgta cgcagttgtc gaacttggtt tgcaaaggga gaaagtagga
     1861 gatctctctt gcgagatgat cccgcatttt cttgaaagct ttgcagaggc tagcagaatt
     1921 accctccacg ttgattgtct gcgaggcaag aatgatcatc accgtagtga gagtgcgttc
     1981 aaggctcttg cggttgccat aagagaagcc acctcgccca atggtaccaa cgatgttccc
     2041 tccaccaaag gtgttcttat gtaggcgaat ttcttatgat ttatgatttt tattattaaa
     2101 taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga
     2161 aaattcttat tcttgagtaa ctctttcctg taggtcaggt tgctttctca ggtatagcat
     2221 gaggtcgctc ttattgacca cacctcagtt tatcattatc aatactcgcc atttcaaaga
     2281 atacgtaaat aattaatagt agtgattttc ctaactttat ttagtcaaaa aattagcctt
     2341 ttaattctgc tgtaacccgt acatgcccaa aatagggggc gggttacaca gaatatataa
     2401 catcgtaggt gtctgggtga acagtttatt cctggcatcc actaaatata atggagcccg
     2461 ctttttaagc tggcatccag aaaaaaaaag aatcccagca ccaaaatatt gttttcttca
     2521 ccaaccatca gttcataggt ccattctctt agcgcaacta cagagaacag gggcacaaac
     2581 aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc tgcctggagt aaatgatgac
     2641 acaaggcaat tgacccacgc atgtatctat ctcattttct tacaccttct attaccttct
     2701 gctctctctg atttggaaaa agctgaaaaa aaaggttgaa accagttccc tgaaattatt
     2761 cccctacttg actaataagt atataaagac ggtaggtatt gattgtaatt ctgtaaatct
     2821 atttcttaaa cttcttaaat tctactttta tagttagtct tttttttagt tttaaaacac
     2881 cagaacttag tttcgacgga ttctagaact agtggatccc ccatcacaaa atgggagacc
     2941 accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggat
     3001 ccgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
     3061 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
     3121 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
     3181 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     3241 catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     3301 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     3361 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     3421 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     3481 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     3541 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     3601 caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
     3661 cagtactgcg atgagtggca gggcggggcg taaacgcgtg gatccggctt actaaaagcc
     3721 agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg
     3781 tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca gtgacagttg
     3841 acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag
     3901 cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca
     3961 ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa
     4021 caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc
     4081 tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc
     4141 tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata
     4201 tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtgtccgtta
     4261 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc
     4321 tgatgttctg gggaatataa ggtctccgga tctggtggag attacaagga tcatgatggt
     4381 gattacaagg atcacgacat cgactacaag gatgacgatg acaagcacca tcatcaccac
     4441 cattgattct atgtcaggct cccttataca cagccagtct gcaggtcgac catagtgact
     4501 ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata
     4561 tattgatatt tatatcattt tacgtttctc gttcagcttt cttgtacaaa gtggtgatgg
     4621 gctgcaggaa ttcgatttca agcttatcga taccgtcgac ctcgagtcat gtaattagtt
     4681 atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt
     4741 agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg
     4801 ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt
     4861 atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgtgacacc
     4921 gattatttaa agctgcagca tacgatatat atacatgtgt atatatgtat acctatgaat
     4981 gtcagtaagt atgtatacga acagtatgat actgaagatg acaaggtaat gcatcattct
     5041 atacgtgtca ttctgaacga ggcgcgcttt ccttttttct ttttgctttt tctttttttt
     5101 tctcttgaac tcgagaaaaa aaatataaaa gagatggagg aacgggaaaa agttagttgt
     5161 ggtgataggt ggcaagtggt attccgtaag aacaacaaga aaagcatttc atattatggc
     5221 tgaactgagc gaacaagtgc aaaatttaag catcaacgac aacaacgaga atggttatgt
     5281 tcctcctcac ttaagaggaa aaccaagaag tgccagaaat aacagtagca actacaataa
     5341 caacaacggc ggctacaacg gtggccgtgg cggtggcagc ttctttagca acaaccgtcg
     5401 tggtggttac ggcaacggtg gtttcttcgg tggaaacaac ggtggcagca gatctaacgg
     5461 ccgttctggt ggtagatgga tcgatggcaa acatgtccca gctccaagaa acgaaaaggc
     5521 cgagatcgcc atatttggtg tccccgagga tccaaatttc caatcttctg gtattaactt
     5581 cgataactac gatgatattc cagtggacgc ctctggtaag gatgttcctg aaccaatcac
     5641 agaatttacc tcacctccat tggacggatt gttattggaa aacatcaaat tggcccgttt
     5701 caccaagcca acacctgtgc aaaaatactc cgtccctatc gttgccaacg gcagagattt
     5761 gatggcctgt gcgcagaccg gttctggtaa gactggtggg tttttattcc cagtgttgtc
     5821 cgaatcattt aagactggac catctcctca accagagtct caaggctcct tttaccaaag
     5881 aaaggcctac ccaactgctg tcattatggc tccagtttaa accatggtca tagctgtttc
     5941 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     6001 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     6061 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6121 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6181 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6241 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6301 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6361 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6421 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6481 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6541 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6601 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6661 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6721 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6781 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6841 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6901 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6961 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     7021 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     7081 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     7141 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7201 ctggccccag tgctgcaatg ataccgcgag atccacgctc accggctcca gatttat
//
