LOCUS       sequence-386861-        6479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for protein expression of 
            9xHis_AtTGA1_HiBit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6479)
  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 6479)
  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: pEPYCdKN0001
FEATURES             Location/Qualifiers
     source          1..6479
                     /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(1332..1356)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1366..1383
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1436..1454)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1450..1497
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1534..1989
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1621..1640)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1831..1852
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2081..2896)
                     /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(2081..2896)
                     /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     2804..2823
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2888..2907
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3018..3606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3507..3526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3760..3777
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3792..3931
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3857..3876)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4017..4039
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4033..4224)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5033..6115)
                     /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     6077..6096
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6116..6193)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6399..6418
                     /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 cttcatgaat tcgacatcga cacattttgt gccaccgaga agagttggta tatacgaacc
      241 tgtccatcaa ttcggtatgt ggggggagag tttcaaaagc aatattagca atgggactat
      301 gaacacacca aaccacataa taataccgaa taatcagaaa ctagacaaca acgtgtcaga
      361 ggatacttcc catggaacag caggaactcc tcacatgttc gatcaagaag cttcaacgtc
      421 tagacatccc gataagatac aaagacggct tgctcaaaac cgcgaggctg ctaggaaaag
      481 tcgcttgcgc aagaaggctt atgttcagca actggaaaca agcaggttga agctaattca
      541 attagagcaa gaactcgatc gtgctagaca acagggattc tatgtaggaa acggaataga
      601 tactaattct ctcggttttt cggaaaccat gaatccaggg attgctgcat ttgaaatgga
      661 atatggacat tgggttgaag aacagaacag acagatatgt gaactaagaa cagttttaca
      721 cggacacatt aacgatatcg agcttcgttc gctagtcgaa aacgccatga aacattactt
      781 tgagcttttc cggatgaaat cgtctgctgc caaagccgat gtcttcttcg tcatgtcagg
      841 gatgtggaga acttcagcag aacgattctt cttatggatt ggcggatttc gaccctccga
      901 tcttctcaag gttcttttgc cacattttga tgtcttgacg gatcaacaac ttctagatgt
      961 atgcaatcta aaacaatcgt gtcagcaagc agaagacgcg ttgactcaag gtatggagaa
     1021 gctgcaacac acccttgcgg actgcgttgc agcgggacaa ctcggtgaag gaagttacat
     1081 tcctcaggtg aattctgcta tggatagatt agaagctttg gtcagtttcg taaatcaggc
     1141 tgatcacttg agacatgaaa cattgcaaca aatgtatcgg atattgacaa cgcgacaagc
     1201 ggctcgagga ttattagctc ttggtgagta ttttcaacgg cttagagcct tgagctcaag
     1261 ttgggcaact cgacatcgtg aaccaacggg ttcggtgagc ggctggcggc tgttcaagaa
     1321 gattagctga aacccagctt tcttgtacaa agtggttgat tcgagcacca ccaccaccac
     1381 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1441 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1501 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1561 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1621 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1681 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1741 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1801 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1861 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1921 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1981 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2041 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     2101 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     2161 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     2221 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     2281 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     2341 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     2401 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     2461 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     2521 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     2581 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     2641 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     2701 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     2761 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     2821 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     2881 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2941 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3001 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3061 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3121 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3181 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3241 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3301 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3361 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3421 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3481 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3541 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3601 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3661 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3721 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3781 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3841 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3901 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3961 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4021 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     4081 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     4141 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     4201 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     4261 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4321 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4381 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4441 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4501 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4561 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4621 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4681 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4741 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4801 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4861 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4921 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4981 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     5041 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5101 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     5161 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     5221 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     5281 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     5341 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5401 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5461 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     5521 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5581 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5641 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5701 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5761 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5821 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5881 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5941 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     6001 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     6061 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     6121 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     6181 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     6241 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     6301 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     6361 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6421 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggat
//
