LOCUS       sequence-386863-        6134 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Gateway destination vector for protein expression of 
            9xHis_AtNAC032_HiBit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6134)
  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 6134)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pEPYCdKN0012
FEATURES             Location/Qualifiers
     source          1..6134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(593..1675)
                     /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     1637..1656
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1676..1753)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1959..1978
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2062..2081
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2062..2080
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2081..2105
                     /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             2120..2142
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2155..2181
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     protein_bind    2197..2221
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3026..3050)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3060..3077
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3130..3148)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3144..3191
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3228..3683
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3315..3334)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3525..3546
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3775..4590)
                     /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(3775..4590)
                     /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     4498..4517
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4582..4601
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4712..5300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5201..5220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5454..5471
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5486..5625
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5551..5570)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5711..5733
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5727..5918)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
       61 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
      121 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
      181 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
      241 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
      301 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
      361 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
      421 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
      481 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
      541 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
      601 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
      661 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
      721 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
      781 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
      841 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
      901 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
      961 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     1021 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     1081 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     1141 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     1201 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     1261 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     1321 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     1381 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     1441 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     1501 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     1561 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     1621 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     1681 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     1741 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     1801 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     1861 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     1921 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     1981 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     2041 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     2101 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gaaacaccac
     2161 caccaccacc accaccacca cggtggtctc gaatcaacaa gtttgtacaa aaaagcaggc
     2221 ttcatgatga aatctggggc tgatttgcaa tttccaccag gatttagatt tcatcctacg
     2281 gatgaggagc tagtcctcat gtatctctgt cgtaaatgcg cgtcgcagcc gatccctgct
     2341 ccgattatca ccgaactcga tttgtaccga tatgatcctt gggaccttcc cgacatggct
     2401 ttgtacggtg aaaaggagtg gtattttttc tcaccaagag atcgaaagta tccaaacggt
     2461 tcaagaccca accgtgcagc tggtactgga tattggaaag ctaccggagc tgataaacca
     2521 ataggtcgtc ctaaaccggt tggtattaag aaggctctag tgttttactc gggaaaacct
     2581 ccaaatggag agaaaaccaa ttggattatg cacgaatacc ggctcgctga cgttgaccgg
     2641 tcggttcgta agaaaaacag tctaagattg gacgattggg tattgtgtcg tatatataac
     2701 aagaaaggtg tcatcgagaa gcgacgaagc gatatcgagg acgggttaaa gcctgtgact
     2761 gacacgtgtc caccggaatc tgtggcgaga ttgatctccg gctcggagca agcggtgtca
     2821 ccggaattca cgtgtagcaa cggtcggttg agtaatgccc ttgattttcc gtttaattac
     2881 gtagatgcca tcgccgataa cgagattgtg tcacggctat tgggcgggaa tcagatgtgg
     2941 tcgacgacgc ttgatccact tgtggttagg cagggaactt tcggttcggt gagcggctgg
     3001 cggctgttca agaagattag ctgaaaccca gctttcttgt acaaagtggt tgattcgagc
     3061 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     3121 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     3181 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     3241 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3301 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3361 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3421 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3481 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3541 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3601 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3661 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     3721 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     3781 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     3841 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     3901 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     3961 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     4021 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     4081 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     4141 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     4201 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     4261 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     4321 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     4381 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     4441 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     4501 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     4561 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     4621 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4681 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4741 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4801 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4861 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4921 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4981 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5041 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5101 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5161 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5221 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5281 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5341 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5401 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5461 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     5521 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5581 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     5641 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5701 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5761 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     5821 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5881 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5941 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     6001 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     6061 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     6121 gtaggtgttc caca
//
