LOCUS       sequence-386127-        6249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression of the His-tagged full length protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6249)
  TITLE     Knapp lab plasmids
REFERENCE   2  (bases 1 to 6249)
  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"
COMMENT     Sequence Label: pNIC28-BIRC7
FEATURES             Location/Qualifiers
     source          1..6249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1114..1131
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1184..1202)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1198..1245
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1282..1737
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1369..1388)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1579..1600
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1829..2644)
                     /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(1829..2644)
                     /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     2552..2571
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2636..2655
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2766..3354
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3255..3274
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3508..3525
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3540..3679
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3605..3624)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3765..3787
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3781..3972)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4781..5863)
                     /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     5825..5844
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5864..5941)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6147..6166
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatggga cctaaagaca gtgccaagtg
      181 cctgcaccgt ggaccacagc cgagccactg ggcagccggt gatggtccca cgcaggagcg
      241 ctgtggaccc cgctctctgg gcagccctgt cctaggcctg gacacctgca gagcctggga
      301 ccacgtggat gggcagatcc tgggccagct gcggcccctg acagaggagg aagaggagga
      361 gggcgccggg gccaccttgt ccagggggcc tgccttcccc ggcatgggct ctgaggagtt
      421 gcgtctggcc tccttctatg actggccgct gactgctgag gtgccacccg agctgctggc
      481 tgctgccggc ttcttccaca caggccatca ggacaaggtg aggtgcttct tctgctatgg
      541 gggcctgcag agctggaagc gcggggacga cccctggacg gagcatgcca agtggttccc
      601 cagctgtcag ttcctgctcc ggtcaaaagg aagagacttt gtccacagtg tgcaggagac
      661 tcactcccag ctgctgggct cctgggaccc gtgggaagaa ccggaagacg cagcccctgt
      721 ggccccctcc gtccctgcct ctgggtaccc tgagctgccc acacccagga gagaggtcca
      781 gtctgaaagt gcccaggagc caggaggggt cagtccagcc gaggcccaga gggcgtggtg
      841 ggttcttgag cccccaggag ccagggatgt ggaggcgcag ctgcggcggc tgcaggagga
      901 gaggacgtgc aaggtgtgcc tggaccgcgc cgtgtccatc gtctttgtgc cgtgcggcca
      961 cctggtctgt gctgagtgtg cccccggcct gcagctgtgc cccatctgca gagcccccgt
     1021 ccgcagccgc gtgcgcacct tcctgtccta acagtaaagg tggatacgga tccgaattcg
     1081 agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
     1141 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1201 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1261 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1321 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1381 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1441 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1501 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1561 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1621 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1681 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     1741 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1801 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     1861 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     1921 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     1981 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     2041 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     2101 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     2161 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     2221 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2281 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     2341 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     2401 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     2461 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     2521 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     2581 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     2641 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     2701 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2761 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2821 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2881 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     2941 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3001 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3061 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3121 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3181 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3241 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3301 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3361 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3421 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3481 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3541 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     3601 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     3661 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3721 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3781 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     3841 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     3901 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     3961 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     4021 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     4081 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     4141 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4201 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4261 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4321 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4381 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4441 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     4501 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     4561 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     4621 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     4681 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     4741 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4801 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4861 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     4921 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     4981 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     5041 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     5101 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     5161 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5221 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5281 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5341 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     5401 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     5461 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     5521 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     5581 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     5641 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     5701 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     5761 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     5821 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     5881 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     5941 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     6001 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     6061 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     6121 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     6181 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     6241 ccgcgaaat
//
