LOCUS       sequence-396568-        7300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MBP-tagged E. coli protein expression destination vector for Golden 
            Gate cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7300)
  AUTHORS   Daffern N, Francino-Urdaniz IM, Baumer ZT, Whitehead TA
  TITLE     Standardizing cassette-based deep mutagenesis by Golden Gate 
            assembly.
  JOURNAL   Biotechnol Bioeng. 2024 Jan;121(1):281-290. doi: 10.1002/bit.28564. 
            Epub 2023 Sep 26.
  PUBMED    37750676
REFERENCE   2  (bases 1 to 7300)
  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: "10.1002/bit.28564";
            journalName: "Biotechnol Bioeng"; date: "2024-01"; volume: "121"; 
            issue: "1"; pages: "281-290"
COMMENT     Sequence Label: pND004
FEATURES             Location/Qualifiers
     source          1..7300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..43)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      39..86
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(202..221)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     412..433
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(662..1477)
                     /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(662..1477)
                     /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     1385..1404
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1469..1488
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1599..2187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2088..2107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2341..2358
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2373..2512
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2438..2457)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2598..2620
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2614..2805)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3614..4696)
                     /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     4658..4677
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4697..4774)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4980..4999
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5087..5106
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5087..5105
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5106..5130
                     /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             5145..5167
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5186..5203
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6430..6441
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             6448..7161
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      7173..7244
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      7260..7287
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
ORIGIN
        1 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
       61 ctaaacgggt cttgaggggt tttttgctgg aactatatcc ggattggccc tcagcgaatg
      121 ggacgcgccc tgtagtaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      181 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      241 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      301 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      361 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      421 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      481 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      541 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
      601 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
      661 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
      721 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
      781 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
      841 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
      901 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
      961 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     1021 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     1081 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     1141 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     1201 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     1261 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     1321 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     1381 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     1441 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     1501 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1561 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1621 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1681 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1741 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1801 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1861 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1921 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1981 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2041 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2101 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2161 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2221 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2281 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2341 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     2401 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     2461 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     2521 cccgccaaca cccgctgacg cgccctgacg ggcttgtttg ctcccggcat ccgcttacag
     2581 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2641 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     2701 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     2761 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     2821 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     2881 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     2941 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     3001 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     3061 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     3121 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     3181 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     3241 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     3301 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     3361 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     3421 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     3481 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     3541 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     3601 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3661 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     3721 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     3781 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     3841 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     3901 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     3961 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     4021 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     4081 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     4141 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     4201 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4261 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     4321 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     4381 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     4441 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     4501 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     4561 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     4621 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     4681 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     4741 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     4801 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     4861 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     4921 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     4981 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     5041 atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaattaat acgactcact
     5101 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     5161 aaggagatat accatgaata gcagccatca tcatcatcat cacagcagcg gcatgaaaat
     5221 cgaagaaggt aaactggtaa tctggattaa cggcgataaa ggctataacg gactcgctga
     5281 agtcggtaag aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa
     5341 actggaagag aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg
     5401 ggcacacgac cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga
     5461 caaagcgttc caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa
     5521 gctgattgct tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct
     5581 gccgaacccg ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa
     5641 aggtaagagc gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc
     5701 tgctgacggg ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg
     5761 cgtggataac gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa
     5821 acacatgaat gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac
     5881 agcgatgacc atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta
     5941 tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct
     6001 gagcgcaggt attaacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa
     6061 ctatctgctg actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt
     6121 agcgctgaag tcttacgagg aagagttggt gaaagatccg cgtattgccg ccactatgga
     6181 aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc
     6241 cgtgcgtact gcggtgatca acgccgccag cggttgagac cgaaagtgaa acgtgatttc
     6301 atgcgtcatt ttgaacattt tgtaaatctt atttaataat gtgtgcggca attcacattt
     6361 aatttatgaa tgttttctta acatcgcggc aactcaagaa acggcaggtt cggatcttag
     6421 ctactagaga aagaggagaa atactagatg cgtaaaggcg aagagctgtt cactggtgtc
     6481 gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc cgtgcgtggc
     6541 gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg tactactggt
     6601 aaactgccgg ttccttggcc gactctggta acgacgctga cttatggtgt tcagtgcttt
     6661 gctcgttatc cggaccatat gaagcagcat gacttcttca agtccgccat gccggaaggc
     6721 tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac gcgtgcggaa
     6781 gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat tgactttaaa
     6841 gaggacggca atatcctggg ccataagctg gaatacaatt ttaacagcca caatgtttac
     6901 atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg ccacaacgtg
     6961 gaggatggca gcgtgcagct ggctgatcac taccagcaaa acactccaat cggtgatggt
     7021 cctgttctgc tgccagacaa tcactatctg agcacgcaaa gcgttctgtc taaagatccg
     7081 aacgagaaac gcgatcatat ggttctgctg gagttcgtaa ccgcagcggg catcacgcat
     7141 ggtatggatg aactgtacaa atgaccaggc atcaaataaa acgaaaggct cagtcgaaag
     7201 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg
     7261 gctcaccttc gggtgggcct ttctgcgttt ataggtctca
//
