LOCUS       sequence-171410-        7382 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This can be used to make MBP-TEV-Cterm with SapI and PstI; and can 
            also make N-term-VMA with NdeI and SapI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7382)
  AUTHORS   Liu J, Chen Q, Rozovsky S
  TITLE     Utilizing Selenocysteine for Expressed Protein Ligation and 
            Bioconjugations.
  JOURNAL   J Am Chem Soc. 2017 Feb 10. doi: 10.1021/jacs.6b10991.
  PUBMED    28186733
REFERENCE   2  (bases 1 to 7382)
  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"; journalName: "J Am Chem 
            Soc. 2017 Feb 10. doi: 10.1021/jacs.6b10991."
COMMENT     Sequence Label: MBP-TEV-VMA Model vector
FEATURES             Location/Qualifiers
     source          1..7382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             81..1163
                     /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     complement(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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)."
     primer_bind     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1540..1557
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     2623..2646
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2774..3592
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="5' region of the intein from the yeast Vma1 
                     subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 5' region
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
     CDS             4048..4059
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4169..4324
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     terminator      4350..4436
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4528..4555
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4583..4673
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4674..4742,4743..5534)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAWQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4892..4911)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5622..6210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6111..6130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6682..6704
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6698..6889)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatttcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cacaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcaattgacc aacaaggacc atagattcat atgaaaatcc atcaccatca ccatcacgaa
     1561 gaaggtaaac tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc
     1621 ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg
     1681 gaagagaaat tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca
     1741 cacgaccgct ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa
     1801 gcgttccagg acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg
     1861 attgcttacc cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg
     1921 aacccgccaa aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt
     1981 aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct
     2041 gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg
     2101 gataacgctg gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac
     2161 atgaatgcag acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg
     2221 atgaccatca acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt
     2281 gtaacggtac tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc
     2341 gcaggtatta acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat
     2401 ctgctgactg atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg
     2461 ctgaagtctt acgaggaaga gttggtgaaa gatccgcgta ttgccgccac tatggaaaac
     2521 gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg
     2581 cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac
     2641 gcgcagacta attcgagctc tgaaaacctg tattttcagt gcggaagagc atgggcaccg
     2701 cttaccgccc ggattggaac cgtctgagtg gcctgacccg tgcgcgtgtg gaaacgtgcg
     2761 gtggtgctct tcctgctttg ccaagggtac caatgtttta atggcggatg ggtctattga
     2821 atgtattgaa aacattgagg ttggtaataa ggtcatgggt aaagatggca gacctcgtga
     2881 ggtaattaaa ttgcccagag gaagagaaac tatgtacagc gtcgtgcaga aaagtcagca
     2941 cagagcccac aaaagtgact caagtcgtga agtgccagaa ttactcaagt ttacgtgtaa
     3001 tgcgacccat gagttggttg ttagaacacc tcgtagtgtc cgccgtttgt ctcgtaccat
     3061 taagggtgtc gaatattttg aagttattac ttttgagatg ggccaaaaga aagcccccga
     3121 cggtagaatt gttgagcttg tcaaggaagt ttcaaagagc tacccaatat ctgaggggcc
     3181 tgagagagcc aacgaattag tagaatccta tagaaaggct tcaaataaag cttattttga
     3241 gtggactatt gaggccagag atctttctct gttgggttcc catgttcgta aagctaccta
     3301 ccagacttac gctccaattc tttatgagaa tgaccacttt ttcgactaca tgcaaaaaag
     3361 taagtttcat ctcaccattg aaggtccaaa agtacttgct tatttacttg gtttatggat
     3421 tggtgatgga ttgtctgaca gggcaacttt ttcggttgat tccagagata cttctttgat
     3481 ggaacgtgtt actgaatatg ctgaaaagtt gaatttgtgc gccgagtata aggacagaaa
     3541 agaaccacaa gttgccaaaa ctgttaattt gtactctaaa gttgtcagag gtaatggtat
     3601 tcgcaataat cttaatactg agaatccatt atgggacgct attgttggct taggattctt
     3661 gaaggacggt gtcaaaaata ttccttcttt cttgtctacg gacaatatcg gtactcgtga
     3721 aacatttctt gctggtctaa ttgattctga tggctatgtt actgatgagc atggtattaa
     3781 agcaacaata aagacaattc atacttctgt cagagatggt ttggtttccc ttgctcgttc
     3841 tttaggctta gtagtctcgg ttaacgcaga acctgctaag gttgacatga atgtcaccaa
     3901 acataaaatt agttatgcta tttatatgtc tggtggagat gttttgctta acgttctttc
     3961 gaagtgtgcc ggctctaaaa aattcaggcc tgctcccgcc gctgcttttg cacgtgagtg
     4021 ccgcggattt tatttcgagt tacaagaatt gaaggaagac gattattatg ggattacttt
     4081 atctgatgat tctgatcatc agtttttgct tggatcccag gttgtcgtcc atgcatgcgg
     4141 tggcctgacc ggtctgaact caggcctcac gacaaatcct ggtgtatccg cttggcaggt
     4201 caacacagct tatactgcgg gacaattggt cacatataac ggcaagacgt ataaatgttt
     4261 gcagccccac acctccttgg caggatggga accatccaac gttcctgcct tgtggcagct
     4321 tcaatgactg caggtaatta aataagcttc aaataaaacg aaaggctcag tcgaaagact
     4381 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     4441 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     4501 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
     4561 ttctacaaac tctttcggtc cgttgtttat ttttctaaat acattcaaat atgtatccgc
     4621 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4681 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4741 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4801 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4861 gtttcccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     4921 acgcctggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4981 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5041 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5101 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5161 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5221 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5281 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5341 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5401 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5461 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5521 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttcctta
     5581 ggactgagcg tcaaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5641 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5701 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5761 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5821 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5881 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5941 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6001 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6061 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6121 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6181 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6241 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6301 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6361 cgcagcgagt cagtgagcga ggaagcggag gcctccgctt cctcgctcac tgactcgcac
     6421 agatgcgtaa ggagaaaata ccgcatcagg cgcacctccg cttcctcgct caccacagat
     6481 gcgtaaggag aaaataccgc atcaggcgct gcgcctgatg cggtattttc tccttacgca
     6541 tctgtgcggt atttcacacc gcatataagg tgcactgtga ctgggtcatg gctgcgcccc
     6601 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     6661 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     6721 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgcagc gattcacaga
     6781 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     6841 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     6901 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     6961 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     7021 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     7081 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     7141 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     7201 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     7261 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     7321 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaaa
     7381 tt
//
