LOCUS       sequence-386855-        7181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for protein expression of 
            9xHis_AtARF18_HiBit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7181)
  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 7181)
  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: pEPYCdKN0022
FEATURES             Location/Qualifiers
     source          1..7181
                     /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(2034..2058)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2068..2085
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2138..2156)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2152..2199
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2236..2691
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2323..2342)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2533..2554
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2783..3598)
                     /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(2783..3598)
                     /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     3506..3525
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3590..3609
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3720..4308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4209..4228
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4462..4479
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4494..4633
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4559..4578)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4719..4741
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4735..4926)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5735..6817)
                     /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     6779..6798
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6818..6895)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7101..7120
                     /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 cttcatggcg agtgttgaag gtgatgatga tttcggaagt tcttcgtcaa ggtcttatca
      241 agatcaacta tacacagagc tatggaaagt ttgtgcaggt ccattagtgg aagttcctcg
      301 tgctcaagag agagttttct acttccctca gggtcacatg gaacaacttg tggcgtcaac
      361 taatcaagga atcaattcag aagaaatacc tgtttttgat cttcctccaa agatactttg
      421 tcgagttctt gatgtcactt taaaggcgga gcatgaaaca gatgaggttt acgctcagat
      481 cacattacaa ccagaggaag atcaaagtga accaacaagt cttgatccac ctattgttgg
      541 accaactaag caagagtttc attcgtttgt taagatttta acggcttcag atacaagcac
      601 tcatggtgga ttctctgttc ttcgtaaaca cgccactgaa tgcttgcctt ctttggatat
      661 gacacaagct actcctactc aagaacttgt gactagagat cttcatggct ttgaatggag
      721 gtttaagcat atattcagag gacaaccacg gaggcatttg cttactacgg gttggagtac
      781 atttgtatcc tcgaaaagac ttgtagctgg agatgctttt gtgttcttga ggggtgagaa
      841 tggggattta cgggttggag tgagacgatt agctcggcat caaagcacaa tgcctacttc
      901 ggttatttca agtcagagca tgcatttggg agttcttgct acagcttctc atgctgtgcg
      961 tacaacaaca atctttgttg tcttttacaa gcctaggata agccaattca tagttggggt
     1021 gaacaagtat atggaagcta taaagcatgg attttctctc ggtacccgat tcagaatgag
     1081 gtttgaagga gaagagtctc ctgagagaat atttactggt acgattgtgg gaagtggaga
     1141 tctatcttca caatggccag cttctaaatg gaggtcattg caggtacaat gggatgagcc
     1201 aacaacagtt caaagaccag ataaagtctc accatgggag atagagcctt tcttggcaac
     1261 ttccccaatt tcaactcctg ctcaacaacc acaatcgaaa tgcaagcggt caagacccat
     1321 cgagccatca gttaaaacac cagccccacc tagtttcttg tacagcctcc ctcagagcca
     1381 agattccatt aatgcatccc ttaaactgtt tcaagatcca tcacttgaga gaatttcagg
     1441 tggatactcc tcaaacaaca gcttcaaacc cgagactcct cctcctccaa cgaattgtag
     1501 ctataggttg tttggatttg atctcacaag caattctcct gctccaatcc ctcaagacaa
     1561 gcaaccgatg gatacttgtg gagctgccaa gtgtcaagaa cccatcactc caacctcaat
     1621 gagtgagcag aagaagcaac aaacatcaag aagtcgaact aaagtgcaaa tgcaaggcat
     1681 tgcggttggt cgtgcggttg atttaacact gttgaaatct tacgatgaac tgattgatga
     1741 gcttgaggag atgtttgaga ttcaaggaca gcttcttgcc cgagacaaat ggatcgttgt
     1801 cttcactgat gatgaaggag atatgatgct tgctggtgat gatccgtgga atgagttttg
     1861 caagatggca aagaagatat ttatatattc gagcgatgag gttaagaaaa tgacaacgaa
     1921 actgaagatt tcttcgtcgt tagagaatga ggaatatggt aatgaatcat tcgaaaatcg
     1981 tagtaggggg ggttcggtga gcggctggcg gctgttcaag aagattagct gaaacccagc
     2041 tttcttgtac aaagtggttg attcgagcac caccaccacc accactgaga tccggctgct
     2101 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2161 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2221 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2281 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2341 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2401 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2461 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2521 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2581 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2641 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     2701 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2761 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     2821 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     2881 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     2941 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3001 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3061 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3121 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3181 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3241 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3301 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3361 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3421 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3481 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3541 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3601 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3661 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3721 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3781 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3841 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3901 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3961 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4021 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4081 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4141 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4201 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4261 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4321 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4381 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4441 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4501 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4561 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4621 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4681 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4741 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4801 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     4861 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     4921 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     4981 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5041 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5101 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5161 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5221 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5281 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5341 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     5401 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     5461 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     5521 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     5581 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     5641 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5701 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5761 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5821 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     5881 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     5941 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     6001 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     6061 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     6121 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     6181 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     6241 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     6301 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     6361 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     6421 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     6481 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     6541 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6601 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6661 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6721 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6781 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     6841 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     6901 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     6961 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     7021 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     7081 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     7141 gcgccggtga tgccggccac gatgcgtccg gcgtagagga t
//
