LOCUS       sequence-380013-        6755 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression of MhCINS protein with a C terminal His-tag and
            transit peptide removed at N terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6755)
  AUTHORS   Liu M-F, Chen J, Goodrich KR, Chiu SK, Pang CC, Scharaschkin T, 
            Saunders RMK
  TITLE     Aerial litter mimicry: A novel form of floral deception mediated by 
            a monoterpene synthase
  JOURNAL   J Ecology 2024; 00, 1–20. doi: 10.1111/1365-2745.14446
REFERENCE   2  (bases 1 to 6755)
  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"; journalName: "J Ecology";
            date: "2024"; volume: " 00, 1–20. doi"; pages: " 
            10.1111/1365-2745.1444"
COMMENT     Sequence Label: pET-MhCINS-6xHis
FEATURES             Location/Qualifiers
     source          1..6755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     954..973
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(993..1070)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1276..1295
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1383..1402
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1383..1401
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1402..1426
                     /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)."
     CDS             3067..3084
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3103..3147
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3185..3203)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3199..3246
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3283..3738
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3370..3389)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3580..3601
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3853..4644,4645..4713))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4476..4495
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4714..4806)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4887..5475
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5376..5395
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5629..5646
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5661..5800
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5726..5745)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5886..5908
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5902..6093)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6665..6755,1..992))
                     /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"
ORIGIN
        1 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
       61 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
      121 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatcca
      181 actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
      241 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
      301 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
      361 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
      421 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
      481 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
      541 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
      601 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
      661 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
      721 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
      781 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
      841 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
      901 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
      961 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     1021 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     1081 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     1141 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     1201 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     1261 ccgatcttcc acatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     1321 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg atcccgcgaa
     1381 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaggagg
     1441 tataatatgc ggagatctgc taattatcga cctagcattt gggactatga ttttatcaaa
     1501 tcactcagtg gtgattacga gtctgatacg tccgcaaaac ggatggaagc actgaaggag
     1561 cagataaggc cgtcgttcca tgggatcaaa gcatcagaaa gcgcggagct aattgacaac
     1621 ctccaaagat tgggaatagg tcatctattt ttaaaggaga tgaaggaagc actagactca
     1681 atgtccatca atgataataa tctggggatg gaaggggacc tccatgctac ggccctcaag
     1741 ttcaggctac ttagacaaca tggttacatg atttcacaag atgtgttcaa taacttcaga
     1801 gatgacgagg gtaattttaa gcaaagcatt ttgatggaca taaagggaac gctgagtcta
     1861 tatgaagctt catatctttc tttccaaggt gaaatcgtct tagataaggc ccaagctttc
     1921 accaaaaaac acctcaagga tctaaaaggg gaagcaaaca caaaccttca aaggttaata
     1981 gatcatgcat tggagcttcc attgcactgg aggtgtccta ggccagagtc caagtggtac
     2041 atagagacat atgaaagaac gcacaacact aatcctgtaa tacttgaatt ggccaagctg
     2101 aacttcaaca tggagcagtc tatcctacaa aaagacttgc aggacgtggc aagatggtgg
     2161 cagaagttag gccttcaaag gaagttgagc ttctgcagag accggatggt ggagtgtttc
     2221 cttttcggtt tgttcatggt agacgatgcc cagttcgaat acggaagaaa agaagtttca
     2281 aaagtaaatc agctagtaac aacgattgat gatatttatg atgtttatgg ttcattggat
     2341 gagctagagc tattcacaaa cgctgttgag agatgggatc tcgaggcaat ggaattactt
     2401 ccagattata tgaagctgtg ctttctagct ctttacaaca ccgctaatga agtagcttat
     2461 cgtgtcttag aggaacaagg atgcaatgca ctgccataca taaagaaagc gtgggcagat
     2521 ttatgtaaag gatatttagt ggaggcaaaa tggtataaca acaaatatac accaacagtc
     2581 gatgagtatc ttgagcattc ttggcgtaca attagtactc atgctgcgct aacctacatt
     2641 tattttgcac gacaacgaga aaatataacg aaagatgaat tatacgtctt agagaattat
     2701 ggagatctta tccgccactg ctgtctgatt ttccggctaa atgatgatct aggaacttca
     2761 aaagatgaga tggctagagg tgattcccca aaagccatcc aatgctacat gcacgaaaag
     2821 ggtatttctg aatcagctgc gcgtgaagaa atagctgttg tgatcagtga gacatggaag
     2881 aagatgaacg aagtatgcat gaattctacg actttctctg aaaactatat caaggctgta
     2941 aagaacactg cacggagtag ccattatgtg taccagtttg gagatgggtt tggagtggta
     3001 tgcaatgaca tgaaggaccg aatcctgggg ctgttggctg aaccaatacc acttgacggc
     3061 ggatcccatc accaccacca tcactaatga ctcgagtctg gtaaagaaac cgctgctgcg
     3121 aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac ctaggctgct
     3181 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     3241 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     3301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     3721 tattaacgtt tacaatttct ggcggcacga tggcatgaga ttatcaaaaa ggatcttcac
     3781 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3841 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3901 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3961 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4021 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4081 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4141 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4201 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4261 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4321 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4381 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4441 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4501 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4561 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4621 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4681 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat catgattgaa
     4741 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4801 aacaaatagg tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4861 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4921 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4981 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     5041 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5101 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5161 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5221 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5281 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5341 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5401 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5461 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5521 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5581 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     5641 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5701 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     5761 atacactcag ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     5821 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5881 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     5941 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     6001 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     6061 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     6121 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     6181 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     6241 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     6301 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     6361 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     6421 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     6481 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     6541 cacgatcatg ctagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     6601 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     6661 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6721 ccaacgcgcg gggagaggcg gtttgcgtat tgggc
//
