LOCUS       sequence-324911-        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        1..78
                     /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             79..1161
                     /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(98..117)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1404..1409,1410..1425,1426..1432)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1440..1456
                     /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     1445..1467
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1526..2626
                     /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     2600..2623
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2675..2686
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2699..3115
                     /codon_start=1
                     /product="bilirubin-binding green fluorescent protein from 
                     Anguilla japonica (Kumagai et al., 2013)"
                     /label=UnaG
                     /translation="MVEKFVGTWKIADSHNFGEYLKAIGAPKELSDGGDATTPTLYISQ
                     KDGDKMTVKIENGPPTFLDTQVKFKLGEEFDEFPSDRRKGVKSVVNLVGEKLVYVQKWD
                     GKETTYVREIKDGKLVVTLTMGDVVAVRSYRRATE"
     CDS             3116..3133
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3172..3258
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3350..3377
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3405..3495
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3496..3564,3565..4356)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3714..3733)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4444..5032
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4933..4952
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5186..5203
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5386..5408
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5402..5593)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
       61 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
      121 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
      181 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
      241 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
      301 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
      361 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
      421 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
      481 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
      541 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
      601 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
      661 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
      721 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
      781 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
      841 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tttcggtagt gggatacgac
      901 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
      961 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     1021 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaataca
     1081 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     1141 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     1201 acaattctca tgtttgacag cttatcatcg actgcacggt gcaccaatgc ttctggcgtc
     1261 aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg
     1321 tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct
     1381 ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat
     1441 tgtgagcgga taacaatttc acacaggaaa cagccagtcc gtttaggtgt tttcacgagc
     1501 aattgaccaa caaggaccat agattatgaa aatcgaagaa ggtaaactgg taatctggat
     1561 taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg agaaagatac
     1621 cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc cacaggttgc
     1681 ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg gtggctacgc
     1741 tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca agctgtatcc
     1801 gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga tcgctgttga
     1861 agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa cctgggaaga
     1921 gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga tgttcaacct
     1981 gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg cgttcaagta
     2041 tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg
     2101 tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc
     2161 catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc
     2221 atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa
     2281 gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc
     2341 gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga
     2401 agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt
     2461 ggtgaaagat ccgcgtattg ccgccactat ggaaaacgcc cagaaaggtg aaatcatgcc
     2521 gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc
     2581 cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt cgagctcgaa
     2641 caacaacaac aataacaata acaacaacct cgggatcgag ggaaggattt cacatatgat
     2701 ggtggaaaag ttcgttggta cctggaaaat cgctgatagc cataattttg gtgaatatct
     2761 gaaggcgatt ggcgctccga aagaactgag cgatggtggc gacgctacca ccccgaccct
     2821 gtacatcagc cagaaagatg gtgacaagat gaccgtgaaa attgaaaatg gtccgccgac
     2881 cttcctggat acccaagtta aatttaagct gggtgaagaa ttcgatgaat ttccgagcga
     2941 ccgtcgtaaa ggtgtgaaga gcgtggttaa cctggttggc gaaaagctgg tgtatgttca
     3001 gaagtgggac ggcaaagaaa ccacctacgt gcgtgaaatc aaagatggta aactggtggt
     3061 gaccctgacc atgggcgacg tggttgcagt tcgtagctat cgtcgtgcga ccgaacatca
     3121 ccatcaccat cactaaggat ccgaattccc tgcaggtaat taaataagct tcaaataaaa
     3181 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     3241 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     3301 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc
     3361 ctgacggatg gcctttttgc gtttctacaa actctttcgg tccgttgttt atttttctaa
     3421 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3481 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3541 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3601 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3661 gagagttttc gccccgaaga acgtttccca atgatgagca cttttaaagt tctgctatgt
     3721 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     3781 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3841 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3901 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3961 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     4021 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     4081 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4141 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4201 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4261 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4321 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4381 atactttaga ttgatttcct taggactgag cgtcaacccc gtagaaaaga tcaaaggatc
     4441 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4501 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4561 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     4621 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4681 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4741 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4801 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4861 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4921 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4981 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5041 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5101 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5161 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     5221 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat aaggtgcact
     5281 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5341 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5401 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     5461 cgtggtcgtg cagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     5521 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     5581 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     5641 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     5701 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     5761 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     5821 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     5881 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     5941 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     6001 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     6061 gccaggaccc aacgctgccc gaaattcc
//
