LOCUS       pET28a(+)-MBP-TE        6735 bp ds-DNA     circular SYN 07-APR-2021
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6735)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Jun 24, 2021 from SnapGene Viewer 5.1.7 to Vector
            NTI(R) format
            https://www.snapgene.com
COMMENT     VNTDATE|945216000|
COMMENT     VNTDBDATE|946166400|
COMMENT     VNTNAME|pET28a(+)-MBP-TEV-Feld3|
COMMENT     VNTAUTHORNAME|Trial User|
FEATURES             Location/Qualifiers
     primer          106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
                     /note="sequence: GGTGATGTCGGCGATATAGG"
     primer          209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
                     /note="sequence: TAATACGACTCACTATAGGG"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
     CDS             335..352
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
     CDS             368..1465
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
     regulatory      1322..1331
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer          1439..1462
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
                     /note="sequence: GATGAAGCCCTGAAAGACGCGCAG"
     CDS             1481..1499
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
     CDS             1505..1801
                     /codon_start=1
                     /label=Feld3
     CDS             1808..1825
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
     primer          complement(1878..1896)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
                     /note="sequence: GCTAGTTATTGCTCAGCGG"
     terminator      1892..1939
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1976..2431
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer          complement(2063..2082)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="sequence: AGGGAAGAAAGCGAAAGGAG"
     primer          2273..2294
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="sequence: GTGGACTCTTGTTCCAAACTGG"
     CDS             complement(2523..3338)
                     /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"
     primer          3246..3265
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
                     /note="sequence: ATCGCGAGCCCATTTATACC"
     primer          3330..3349
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
                     /note="sequence: ATGGCTCATAACACCCCTTG"
     rep_origin      3460..4048
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer          3949..3968
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="sequence: GGGAAACGCCTGGTATCTTT"
     primer          4202..4219
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="sequence: AGCGAGTCAGTGAGCGAG"
     misc_feature    4234..4373
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer          complement(4299..4318)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /note="sequence: CGGCATCAGAGCAGATTGTA"
     primer          4459..4481
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
                     /note="sequence: CCGGGAGCTGCATGTGTCAGAGG"
     CDS             complement(4475..4666)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5475..6557)
                     /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          6519..6538
                     /label=LacI-R
                     /note="LacI, reverse primer"
                     /note="sequence: GGCATACTCTGCGACATCGT"
     promoter        complement(6558..6635)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatggc
      361 tagcggtaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
      421 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
      481 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
      541 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
      601 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
      661 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
      721 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
      781 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
      841 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
      901 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
      961 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
     1021 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
     1081 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
     1141 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcgaaaga
     1201 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
     1261 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc
     1321 cgccaccatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
     1381 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga
     1441 tgaagccctg aaagacgcgc agactcgtat caccaagggt gaaaatctat acttccaagg
     1501 atccatgatt cctggtggtc tgagcgaagc aaaaccggca acaccggaaa ttcaagaaat
     1561 tgccaatgaa gttaaaccgc agctggaaga aaaaaccaat gaaacctatc agaaattcga
     1621 ggccatcgag tataaaaccc aggttgttgc aggtatcaac tactatatca aagtccaggt
     1681 ggatgataac cgctacatcc acattaaagt gtttaaaggt ctgccggttc aggatagcag
     1741 tctgaccctg accggttatc agaccggtaa aagcgaagat gatgaactga ccggctttta
     1801 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     1861 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1921 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     1981 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2041 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2101 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2161 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2221 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2281 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2341 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2401 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     2461 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     2521 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2581 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2641 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2701 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     2761 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2821 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2881 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     2941 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     3001 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     3061 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     3121 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     3181 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     3241 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     3301 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     3361 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     3421 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3481 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3541 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3601 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3661 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3721 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3781 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3841 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3901 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3961 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4021 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4081 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4141 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4201 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     4261 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     4321 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     4381 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4441 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4501 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4561 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4621 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4681 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4741 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4801 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4861 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4921 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4981 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     5041 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     5101 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     5161 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     5221 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     5281 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     5341 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     5401 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     5461 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5521 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     5581 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     5641 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     5701 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     5761 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     5821 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     5881 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     5941 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     6001 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     6061 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     6121 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     6181 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     6241 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     6301 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     6361 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6421 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     6481 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     6541 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     6601 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     6661 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     6721 aaggaatggt gcatg
//
