LOCUS       sequence-36280-e        7200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7200)
  AUTHORS   Diestra E, Fontana J, Guichard P, Marco S, Risco C
  TITLE     Visualization of proteins in intact cells with a clonable tag for 
            electron microscopy.
  JOURNAL   J Struct Biol. 2009 Mar;165(3):157-68. Epub 2008 Dec 10.
  PUBMED    19114107
REFERENCE   2  (bases 1 to 7200)
  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 Struct 
            Biol."; date: "2009-03"; volume: "165(3)"; pages: "157-68. Epub 2008
            Dec 10"
COMMENT     Sequence Label: pMAL-c2x-MT3
FEATURES             Location/Qualifiers
     source          1..7200
                     /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             1528..2628
                     /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."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2602..2625
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2677..2688
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3290..3307)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3290..3306)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3299..3321)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3485..3502)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3655..3741
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3833..3860
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3880..3971
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3972..4040,4041..4832)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4190..4209)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4874..5387)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5069..5090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5281..5300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5498..6086
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5987..6006
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6240..6257
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6272..6414
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6340..6359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6500..6522
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6516..6707)
                     /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 tatctcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc 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 gcacttcacc aacaaggacc atagcatatg aaaatcgaag aaggtaaact ggtaatctgg
     1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     2461 ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat ttcaagatat
     2701 accatggacc ccaactgctc ctgctccacc ggcggctcct gcacttgcac cagctcctgc
     2761 gcctgcaaga actgcaagtg cacctcctgc aagaagagct gctgctcctg ctgtcccgtg
     2821 ggctgctcca aatgtgccca gggctgtgtc tgcaaaggcg ccgcggacaa gtgcacgtgc
     2881 tgtgacatgg accccaactg ctcctgctcc accggcggct cctgcacttg caccagctcc
     2941 tgcgcctgca agaactgcaa gtgcacctcc tgcaagaaga gctgctgctc ctgctgtccc
     3001 gtgggctgct ccaaatgtgc ccagggctgt gtctgcaaag gcgccgcgga caagtgcacg
     3061 tgctgtgaca tggaccccaa ctgctcctgc tccaccggcg gctcctgcac ttgcaccagc
     3121 tcctgcgcct gcaagaactg caagtgcacc tcctgcaaga agagctgctg ctcctgctgt
     3181 cccgtgggct gctccaaatg tgcccagggc tgtgtctgca aaggcgccgc ggacaagtgc
     3241 acgtgctgtg cctaggatcc tctagagtcg acctgcaggc aagcttggca ctggccgtcg
     3301 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     3361 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     3421 agttgcgcag cctgaatggc gaatggcagc ttggctgttt tggcggatga gataagattt
     3481 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg
     3541 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
     3601 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
     3661 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     3721 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
     3781 ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc
     3841 atcctgacgg atggcctttt tgcgtttcta caaactcttt ttgtttattt ttctaaatac
     3901 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3961 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4021 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4081 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4141 gttttcgccc cgaagaacgt tctccaatga tgagcacttt taaagttctg ctatgtggcg
     4201 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4261 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4321 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4381 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4441 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4501 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4561 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4621 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4681 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     4741 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     4801 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4861 tttagattga tttaccccgg ttgataatca gaaaagcccc aaaaacagga agattgtata
     4921 agcaaatatt taaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     4981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     5041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     5101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     5161 accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     5221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     5281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     5341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtaaa aggatctagg
     5401 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5461 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5521 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5581 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5641 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5701 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5761 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5821 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5881 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5941 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6001 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6061 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6121 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6181 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     6241 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     6301 tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct ctgatgccgc
     6361 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     6421 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6481 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6541 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgcagcga ttcacagatg
     6601 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     6661 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     6721 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     6781 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     6841 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     6901 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     6961 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     7021 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     7081 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     7141 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgaaatt
//
