LOCUS       sequence-316709-        6088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid with UnaG coding sequence cloned into it. UnaG gene codon 
            optimized for E. coli and B. theta. Expresses UnaG with N-term 
            maltose-binding protein (MBP)-tag and C-term His-tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6088)
  AUTHORS   Chia HE, Zuo T, Koropatkin NM, Marsh ENG, Biteen JS
  TITLE     Imaging living obligate anaerobic bacteria with bilin-binding 
            fluorescent proteins.
  JOURNAL   Curr Res Microb Sci. 2020 Sep;1:1-6. doi: 
            10.1016/j.crmicr.2020.04.001. Epub 2020 May 11.
  PUBMED    33313576
REFERENCE   2  (bases 1 to 6088)
  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: "Curr Res 
            Microb Sci."; date: "2020-09"; volume: "1:1-6. doi"; pages: " 
            10.1016/j.crmicr.2020.04.001. Epub 2020 May 11"
COMMENT     Sequence Label: pMAL-c5x_UnaG
FEATURES             Location/Qualifiers
     source          1..6088
                     /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
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     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"
     CDS             2701..3117
                     /codon_start=1
                     /product="bilirubin-binding green fluorescent protein from 
                     Anguilla japonica (Kumagai et al., 2013)"
                     /label=UnaG
                     /translation="MVEKFVGTWKIADSHNFGEYLKAIGAPKELSDGGDATTPTLYISQ
                     KDGDKMTVKIENGPPTFLDTQVKFKLGEEFDEFPSDRRKGVKSVVNLVGEKLVYVQKWD
                     GKETTYVREIKDGKLVVTLTMGDVVAVRSYRRATE"
     CDS             3118..3135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3174..3260
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3352..3379
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3407..3497
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3498..3566,3567..4358)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRHIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3716..3735)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4446..5034
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4935..4954
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5188..5205
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5388..5410
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5404..5595)
                     /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 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 gcaattgacc aacaaggacc atagattatg 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 ttggtgaaag atccgcgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat ttcacatatg
     2701 atggtggaaa agttcgttgg tacctggaaa atcgctgata gccataattt tggtgaatat
     2761 ctgaaggcga ttggcgctcc gaaagaactg agcgatggtg gcgacgctac caccccgacc
     2821 ctgtacatca gccagaaaga tggtgacaag atgaccgtga aaattgaaaa tggtccgccg
     2881 accttcctgg atacccaagt taaatttaag ctgggtgaag aattcgatga atttccgagc
     2941 gaccgtcgta aaggtgtgaa gagcgtggtt aacctggttg gcgaaaagct ggtgtatgtt
     3001 cagaagtggg acggcaaaga aaccacctac gtgcgtgaaa tcaaagatgg taaactggtg
     3061 gtgaccctga ccatgggcga cgtggttgca gttcgtagct atcgtcgtgc gaccgaacat
     3121 caccatcacc atcactaagg atccgaattc cctgcaggta attaaataag cttcaaataa
     3181 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     3241 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     3301 gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca
     3361 tcctgacgga tggccttttt gcgtttctac aaactctttc ggtccgttgt ttatttttct
     3421 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3481 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3541 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3601 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3661 ttgagagttt tcgccccgaa gaacgtttcc caatgatgag cacttttaaa gttctgctat
     3721 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cacatacact
     3781 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3841 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3901 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3961 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4021 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4081 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4141 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4201 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4261 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4321 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4381 atatacttta gattgatttc cttaggactg agcgtcaacc ccgtagaaaa gatcaaagga
     4441 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4501 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4561 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4621 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4681 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4741 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4801 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4861 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4921 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4981 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5041 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5101 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5161 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     5221 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat ataaggtgca
     5281 ctgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     5341 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5401 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     5461 agcgtggtcg tgcagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     5521 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     5581 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     5641 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     5701 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     5761 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     5821 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5881 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5941 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     6001 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     6061 tggccaggac ccaacgctgc ccgaaatt
//
