LOCUS       sequence-386844-        8252 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for protein expression of 
            9xHis_AtNLP7_HiBit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8252)
  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 8252)
  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: pEPYCdKN0010
FEATURES             Location/Qualifiers
     source          1..8252
                     /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(3105..3129)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3139..3156
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3209..3227)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3223..3270
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3307..3762
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3394..3413)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3604..3625
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3854..4669)
                     /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(3854..4669)
                     /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     4577..4596
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4661..4680
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4791..5379
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5280..5299
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5533..5550
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5565..5704
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5630..5649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5790..5812
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5806..5997)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6806..7888)
                     /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     7850..7869
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7889..7966)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8172..8191
                     /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 cttcatgtgc gagcccgatg ataattccgc tagaaacggc gtcactactc aaccttcgag
      241 gtcaagggag cttctaatgg atgttgacga cttagatctt gacggttcat ggccactaga
      301 tcaaatccct tacttatcct catcgaatcg catgatttct ccgatttttg tctcctcttc
      361 ctctgagcag ccttgctcgc ctctctgggc tttctccgac ggtggaggaa atggttttca
      421 ccacgcaacc tccggtggcg atgatgagaa gatcagctct gtctccggtg ttccttcttt
      481 ccgtctcgcc gagtatcctc tcttcctccc ttactcttct ccatcagcag ctgagaacac
      541 aacagagaag cataacagtt tccagtttcc gtctccattg atgagcctag tcccaccaga
      601 gaacacagac aactactgtg tgatcaaaga gaggatgact caggcgcttc gatacttcaa
      661 agaatcaacc gaacaacacg ttttggctca ggtctgggct cctgtgagaa agaatggtcg
      721 tgatttgctg acgactttgg gtcaaccttt tgttcttaat cctaatggta atgggcttaa
      781 tcaatacagg atgatctctc tcacatatat gttttctgtg gatagtgaaa gtgacgtaga
      841 gctcggactc ccgggtcgag ttttccgtca gaaattgcct gaatggactc caaatgttca
      901 gtactattcc agcaaagaat tctcgcggct tgatcacgcc ttgcactaca acgtgcgtgg
      961 tacactggcc ttgcctgtct ttaatccctc tggtcagtcc tgcataggtg ttgtggaact
     1021 tataatgacc tcagagaaga ttcactatgc acccgaagtg gacaaagttt gcaaagccct
     1081 tgaggcggta aatctgaaaa gctcggaaat acttgatcac caaacaacac agatatgcaa
     1141 tgagagtcgc caaaacgcgc ttgctgagat tctcgaagtg ctgacagttg tatgtgaaac
     1201 ccataacttg cctctcgctc agacttgggt tccatgtcag catgggagcg ttcttgccaa
     1261 tggtggcggt ctaaagaaaa actgcaccag ctttgacggt agctgcatgg gtcaaatctg
     1321 catgtctaca accgacatgg cctgctatgt cgtggatgct catgtctggg gctttagaga
     1381 tgcctgtctt gaacaccatc tccagaaagg ccagggagtc gctggacgag cttttctcaa
     1441 tggtggctca tgtttctgca gagacatcac caagttctgc aaaacgcagt acccactagt
     1501 ccattatgcg ctcatgttca agttgaccac ttgttttgca atatctctcc agagctctta
     1561 cacgggcgac gacagttaca ttcttgaatt ttttcttcct tcgagtataa cagacgacca
     1621 agagcaagat ttgctgttgg gttctatttt ggtgacaatg aaagaacatt ttcagagtct
     1681 gagggttgca tctggggttg actttggtga agatgacgac aaattgtctt tcgagatcat
     1741 ccaagcatta ccggacaaga aggttcattc aaaaatagaa tccattcgag ttcccttttc
     1801 tggttttaag tcaaatgcaa cagagacgat gttgattcct cagcctgtgg ttcagtcttc
     1861 tgatccagta aatgagaaaa tcaacgtggc cactgttaac ggtgtggtta aggagaagaa
     1921 gaaaacagag aaaaagcgtg ggaagactga gaaaacaatc agtctagatg tacttcagca
     1981 gtatttcact ggaagtctca aagacgctgc aaagagccta ggagtttgcc cgacgacaat
     2041 gaagcgaatt tgcaggcaac acggaatctc gcggtggcca tcgaggaaga tcaagaaagt
     2101 gaatcgttca atcacaaagc tgaaacgagt catcgaatct gttcaaggta ctgatggagg
     2161 cctcgacctg acttccatgg ccgttagttc catcccttgg acacacggtc aaacatcagc
     2221 acagccacta aactcaccca atggttccaa accacctgag ctaccaaaca ccaataattc
     2281 acctaaccat tggtcaagtg atcacagtcc gaacgagcca aatggttcgc ctgagttacc
     2341 accaagcaat ggtcacaagc gatcacgaac ggtggatgag agcgctggga ctccaacctc
     2401 tcatggctca tgtgacggta accaattaga tgaaccgaaa gtcccaaatc aagatccgct
     2461 tttcacggtt ggtggatcac ccgggctcct ttttccacct tattctagag atcatgatgt
     2521 atctgcagct tccttcgcaa tgccgaacag gcttcttggt tctatagacc atttccgagg
     2581 aatgctcatt gaagacgctg gaagttcaaa agatctgaga aatctctgcc ccactgcagc
     2641 atttgacgat aagtttcaag acacaaactg gatgaacaat gataataata gcaacaacaa
     2701 cttatacgct cccccaaagg aagaggccat tgcaaatgtt gcatgcgaac catcaggctc
     2761 agaaatgaga acggtaacaa tcaaagcaag ttacaaagac gacataatac ggttcagaat
     2821 atcctcgggt tcaggtataa tggaattgaa ggatgaagtg gctaagaggc tgaaagttga
     2881 tgcaggaacg ttcgatatca agtatcttga cgatgataac gaatgggttt taatagcttg
     2941 tgatgctgat cttcaagaat gtctcgagat ccctagatcc tcccgcacga aaatcgtaag
     3001 gctcttagtt catgatgtaa cgacaaatct agggagctcc tgcgagagca ctggagaatt
     3061 gggttcggtg agcggctggc ggctgttcaa gaagattagc tgaaacccag ctttcttgta
     3121 caaagtggtt gattcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     3181 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     3241 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     3301 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3361 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3421 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3481 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3541 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3601 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3661 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3721 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     3781 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3841 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     3901 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     3961 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     4021 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     4081 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     4141 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     4201 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     4261 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     4321 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     4381 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     4441 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     4501 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     4561 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     4621 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     4681 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     4741 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4801 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4861 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4921 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4981 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5041 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5101 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5161 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5221 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5281 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5341 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5401 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5461 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5521 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     5581 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     5641 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     5701 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5761 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5821 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5881 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5941 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     6001 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     6061 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     6121 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     6181 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     6241 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     6301 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     6361 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     6421 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     6481 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     6541 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     6601 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     6661 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     6721 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     6781 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6841 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     6901 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     6961 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     7021 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     7081 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     7141 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     7201 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     7261 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     7321 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     7381 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     7441 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     7501 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     7561 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     7621 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     7681 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     7741 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     7801 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     7861 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     7921 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     7981 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     8041 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     8101 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     8161 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     8221 atgccggcca cgatgcgtcc ggcgtagagg at
//
