LOCUS       sequence-386862-        6365 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for protein expression of 
            9xHis_AtTGA2_HiBit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6365)
  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 6365)
  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: pEPYCdKN0002
FEATURES             Location/Qualifiers
     source          1..6365
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     91..113
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(107..298)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1107..2189)
                     /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     2151..2170
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2190..2267)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2473..2492
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2576..2595
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2576..2594
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2595..2619
                     /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             2634..2656
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2669..2695
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     protein_bind    2711..2735
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3771..3795)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3805..3822
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3875..3893)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3889..3936
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3973..4428
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4060..4079)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4270..4291
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4520..5335)
                     /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(4520..5335)
                     /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     5243..5262
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5327..5346
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5457..6045
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5946..5965
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6199..6216
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6231..6365,1..5)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6296..6315)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
       61 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
      121 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
      181 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
      241 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
      301 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
      361 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
      421 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
      481 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
      541 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
      601 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
      661 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
      721 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
      781 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
      841 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
      901 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
      961 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     1021 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     1081 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     1141 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     1201 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     1261 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     1321 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     1381 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     1441 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     1501 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     1561 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     1621 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     1681 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     1741 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     1801 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     1861 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     1921 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     1981 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     2041 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     2101 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     2161 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     2221 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     2281 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     2341 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     2401 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     2461 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     2521 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     2581 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     2641 aactttaaga aggagatata ccatgaaaca ccaccaccac caccaccacc accacggtgg
     2701 tctcgaatca acaagtttgt acaaaaaagc aggcttcatg gctgatacca gtccgagaac
     2761 tgatgtctca acagatgacg acacagatca tcctgatctt gggtcggagg gagcactagt
     2821 gaatactgct gcttctgatt cgagtgaccg atcgaaggga aagatggatc aaaagactct
     2881 tcgtaggctt gctcaaaacc gtgaggcagc aaggaaaagc agattgagga agaaggctta
     2941 tgttcagcag ctagagaaca gccgcttgaa actaacccag cttgagcagg agctgcaaag
     3001 agcaagacag cagggcgtat tcatttcagg cacaggtgac caggcccatt ctactggtgg
     3061 aaatggtgct ttggcgtttg atgctgaaca ttcacggtgg ttggaagaaa agaacaagca
     3121 aatgaacgag ctgaggtctg ctctgaatgc gcatgcaggt gattctgagc ttcgaataat
     3181 agtcgatggt gtgatggctc actatgagga gcttttcagg ataaagagca atgcagctaa
     3241 gaatgatgtc tttcacttgc tatctggcat gtggaaaaca ccagctgaga gatgtttctt
     3301 gtggctcggt ggatttcgtt catccgaact tctaaagctt ctggcgaatc agttggagcc
     3361 aatgacagag agacagttga tgggcataaa taacctgcaa cagacatcgc agcaggctga
     3421 agatgctttg tctcaaggga tggagagctt acaacagtca ctagctgata ctttatcgag
     3481 cgggactctt ggttcaagtt catcagggaa tgtcgcaagc tacatgggtc agatggccat
     3541 ggcaatggga aagttaggta cactcgaagg atttatccgc caggctgata atttgagact
     3601 acaaacattg caacagatga taagagtatt aacaacgaga cagtcagcac gtgctctact
     3661 tgcaatacac gattacttct cacggctacg agctctaagc tccttatggc ttgctcgacc
     3721 cagagagggt tcggtgagcg gctggcggct gttcaagaag attagctgaa acccagcttt
     3781 cttgtacaaa gtggttgatt cgagcaccac caccaccacc actgagatcc ggctgctaac
     3841 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     3901 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     3961 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4021 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4081 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4141 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4201 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4261 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4321 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4381 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     4441 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4501 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     4561 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     4621 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     4681 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     4741 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     4801 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     4861 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     4921 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     4981 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     5041 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     5101 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     5161 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     5221 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     5281 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     5341 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     5401 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5461 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5521 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5581 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5641 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5701 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5761 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5821 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5881 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5941 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6001 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6061 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6121 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6181 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     6241 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     6301 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     6361 catgg
//
