LOCUS       sequence-386945-        7898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for protein expression of 
            9xHis_AtNLP6_HiBit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7898)
  AUTHORS   Cai YM, Witham S, Patron NJ
  TITLE     Tuning Plant Promoters Using a Simple Split Luciferase Method to 
            Assess Transcription Factor-DNA Interactions.
  JOURNAL   ACS Synth Biol. 2023 Nov 17;12(11):3482-3486. doi: 
            10.1021/acssynbio.3c00094. Epub 2023 Oct 19.
  PUBMED    37856867
REFERENCE   2  (bases 1 to 7898)
  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: 
            "10.1021/acssynbio.3c00094"; journalName: "ACS Synth Biol"; date: 
            "2023-11-17- 17"; volume: "12"; issue: "11"; pages: "3482-3486"
COMMENT     Sequence Label: pEPYCdKN0009
FEATURES             Location/Qualifiers
     source          1..7898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..42
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        23..41
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    42..66
                     /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)."
     RBS             81..103
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             116..142
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     protein_bind    158..182
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2751..2775)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2785..2802
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2855..2873)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2869..2916
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2953..3408
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3040..3059)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3250..3271
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3500..4315)
                     /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             complement(3500..4315)
                     /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     4223..4242
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4307..4326
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4437..5025
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4926..4945
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5179..5196
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5211..5350
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5276..5295)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5436..5458
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5452..5643)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6452..7534)
                     /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     7496..7515
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7535..7612)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7818..7837
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
       61 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tgaaacacca
      121 ccaccaccac caccaccacc acggtggtct cgaatcaaca agtttgtaca aaaaagcagg
      181 cttcatggaa cttgacgact tggatctcag cggttcttgg ccgttagatc agatcacttt
      241 tgcttcaaat ttcaagtctc ctgtcatttt ctcttcctcc gaacaacctt tctctcctct
      301 ctggtccttc tccgaaactt ctggagatgt cggcggcgag ctttactccg ccgcagtagc
      361 tcccactcgc ttcaccgatt attccgtctt actcgctagt tctgaatctg aaaccacgac
      421 caaggagaat aatcaggttc cttctccgtc atggggtata atgcctttgg agaatccaga
      481 tagttactgt gccatcaaag ccaagatgac tcaagcgctt agatacttca aggaatctac
      541 tggacaacaa catgttcttg ctcaggtttg ggcgcctgtc aaaaaccgtg gtcgatatgt
      601 tcttacaact tcaggacagc catttgttct tggtcccaat agtaatggcc ttaatcaata
      661 caggatggtt tcattgacgt atatgttttc ccttgatggt gaacgggacg gagagctcgg
      721 gcttcctggt cgcgttttca ggaaaaagtt acctgagtgg actcctaatg tgcagtatta
      781 ctccagcaaa gagttttcaa ggcttggtca cgccttgcac tataatgttc agggaacttt
      841 ggctttgcct gtttttgaac catccaggca actctgtgta ggagttgtcg agcttattat
      901 gacttctccg aagattaact atgctcctga ggttgaaaaa gtttgtaaag cgcttgaggc
      961 cgtgaatctg aaaacttcgg aaatattgaa ccacgagact actcagatct gcaacgaggg
     1021 tcgccaaaat gcgttagctg agattttgga gatactgaca gtggtgtgcg aaacctacaa
     1081 actgccttta gcccagacat gggtcccttg caggcatcgc agtgttctag cctttggagg
     1141 tggttttaag aaaagctgtt cgagcttcga tggaagctgt atggggaaag tctgtatgtc
     1201 tactagtgat ttagcggttt atgttgttga tgctcatgtt tggggtttcc gagacgcctg
     1261 tgctgaacac catctgcaga agggacaagg tgttgctggg agggcgtttc agtctggaaa
     1321 tctatgcttc tgtagagatg ttactcggtt ctgcaaaacc gactacccat tagtacacta
     1381 tgcacggatg ttcaagttga cgagctgttt cgcggtctgc ttaaagagca catatacagg
     1441 ggatgatgag tacgttcttg aattctttct tcctccagcc atcacagaca agagtgagca
     1501 agattgtcta ttgggttctc tgcttcagac aatgaagcag cactattcga gtctcaaggt
     1561 tgtctctgaa actgaactct gtgaaaacaa catgtctctt gaagtcgtgg aagcttcgga
     1621 ggacggaatg gtttactcga aacttgaacc aatcaggata catcatccag cacaaatctc
     1681 taaggattac cttgagctca acgcacccga acagaaggtg agcttaaact ctgactttat
     1741 ggagaataat gaagttgatg atggggttga gaggttccaa acgcttgatc ctataccaga
     1801 ggccaaaaca gtgaaaaaat cagaaaggaa gcgtgggaaa acagagaaaa cgatcagtct
     1861 agaagtgctt caacaatatt ttgctggtag tctaaaagat gcagcaaaga gcctcggagt
     1921 ttgcccaaca acaatgaaac gcatttgccg acaacacggg atctcacggt ggccatctcg
     1981 aaaaatcaac aaggtgaacc gatctctcac acgacttaaa catgttatag actcggttca
     2041 aggtgctgat ggatcactca acttgacgtc cctctctcct cgcccttggc ctcaccaaat
     2101 ccctccaatc gatatccaat tagcaaagaa ctgccctcca acttctacat ctcccctaag
     2161 taatctccaa gatgtcaaaa tcgaaaacag agatgcagaa gacagtgcag gttcatcaac
     2221 atcgcgtgct tcttgcaaag tgaatcctat ttgcgagacg cgttttcggc ttcctacaca
     2281 caaccaagaa ccatccagac aagtggcctt ggacgattcg gatagtagct ctaaaaacat
     2341 gacaaatttt tgggcacact taacctgcca agatacagct tctccaacaa ttctccaaca
     2401 taagctcgtg agtataaagg caacgtacag agaagacatc atcaggttca agatctcacc
     2461 agagtcggta agcattacgg aacttaagca gcaagtggcc aaaaggctga agctcgaaac
     2521 ggcagccttt gagctcaagt atctagacga cgatagagaa tgggtgtccg tctcatgcga
     2581 tgctgacctc agcgaatgcc tcgacacgtc tgcagcaaaa gcgaacacac tgaggctctc
     2641 agtacatgac gtgactttta actttgggag ctcatgcgaa agctcagagg aaactatgat
     2701 gtgcttgggt tcggtgagcg gctggcggct gttcaagaag attagctgaa acccagcttt
     2761 cttgtacaaa gtggttgatt cgagcaccac caccaccacc actgagatcc ggctgctaac
     2821 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     2881 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     2941 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3001 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3061 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3121 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3181 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3241 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3301 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3361 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     3421 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3481 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     3541 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     3601 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     3661 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     3721 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     3781 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     3841 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     3901 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     3961 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     4021 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     4081 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     4141 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     4201 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     4261 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     4321 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     4381 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4441 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4501 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4561 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4621 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4681 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4741 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4801 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4861 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4921 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4981 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5041 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5101 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     5161 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     5221 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     5281 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     5341 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5401 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5461 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     5521 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     5581 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     5641 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     5701 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     5761 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     5821 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     5881 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     5941 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     6001 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     6061 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     6121 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     6181 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     6241 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     6301 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     6361 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     6421 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6481 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6541 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     6601 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     6661 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     6721 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     6781 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     6841 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     6901 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     6961 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     7021 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     7081 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     7141 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     7201 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     7261 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     7321 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     7381 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     7441 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     7501 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     7561 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     7621 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     7681 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     7741 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     7801 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     7861 ccggtgatgc cggccacgat gcgtccggcg tagaggat
//
