LOCUS       sequence-483097-        8562 bp    DNA     circular SYN 13-AUG-2025
DEFINITION  Upregulation of mgdh with tufB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8562)
  AUTHORS   Schmitz AM, Pian B, Marecos S, Wu M, Holycross M, Gazel E, Reid MC, 
            Barstow B
  TITLE     High Efficiency Rare Earth Element Biomining with Systems Biology 
            Guided Engineering of Gluconobacter oxydans
  JOURNAL   bioRxiv 2023.02.09.527855; doi: 10.1101/2023.02.09.527855
REFERENCE   2  (bases 1 to 8562)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 13, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2023.02.09.527855; doi: 10.1101/2023.02.09.527855"
COMMENT     Sequence Label: pKOS6b-URtufB
FEATURES             Location/Qualifiers
     source          1..8562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2933..2950)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2933..2949)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2942..2964)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3454..4248
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3508..3527)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4118..4137
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             5800..7080
                     /codon_start=1
                     /gene="codA"
                     /product="E. coli cytosine deaminase"
                     /label=codA
                     /note="confers sensitivity to 5-fluorocytosine"
                     /translation="SNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPITENS
                     LDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVKQRA
                     WQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSYP
                     NGEALLEEALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDEQ
                     SRFVETVAALAHHEGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVNIH
                     LQGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQL
                     MGYGQINDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSVR
                     GGKVIASTQPAQTTVYLEQPEAIDYKR"
     oriT            7485..7594
                     /note="incP origin of transfer"
     promoter        complement(7829..7919)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        complement(8113..8203)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga catgattacg aattcgagct cggtacccgg
     1021 ggatcctgcg cggaaatcac gcttcttgtt gcgacggtca cgatacgcat aacgcagcgc
     1081 cttctcgacg cgctccagag cgatacggta gttcgtagac gaacggccgc gatagccctt
     1141 ggcgagctcg agaaccttct tgtgacgggc gtgcgacgtt acgccccgct tgacacgtgc
     1201 catttacctt atctcctcag agcccgtagg gcgcccactg cttgattgtc ttcgcatcca
     1261 tgtccgtcat ggtctgggga ccacggttgg tgcgcttcat cttctgcgga cggttgataa
     1321 ggccatggcg cttgttgccc gggccgcaca tcactttgcc ggtcgcggtg atcttgaacc
     1381 gcttcttgac cgacgacttg gtcttcatct tgggcatttc ggtctccctc gccgtaaacg
     1441 attggtccct cacacagtaa ggactctgca cggccgggca tgccactcgt ggcccggacg
     1501 cgtgcgcgga agcggtgtcc ttatcgtaag cccaagtggc ctgcaagctc attattttct
     1561 tgaacgctca atcataagtc cttcctttcc tcaagtcacg acgacgtgaa agtcctcaca
     1621 gtgacgtgag caagatgaac cttctgacat cttgaccctt gacggtcatc catgtgcttt
     1681 gtccggaagt caccgtgatg catcagaaca acagatccag gaacatcccg aaggcatcgt
     1741 ttacggtgct ctcgaagtca tgcgccgtcg cggcggcacg actgcagatc cggtggccat
     1801 gttccactcg gctctggaca acgtgaagcc tgcggttgaa gtccgctcgc gtcgcgtcgg
     1861 tggcgcaacc tatcaggttc cggttgaagt ccgcgccgag cgccgtcagg ctctcgcgat
     1921 ccgctggctg atcgatgcct cccgcaagcg tggcgagaac acgatgcagg agcgtctgtc
     1981 caacgaactg atggatgcgg tcaacaaccg tggttccgct gtcaagaagc gcgaagacac
     2041 gcaccgcatg gctgaagcca acaaggcatt cagccactat cgctggtaat cagaaccggt
     2101 taaagggctt ccggcagtgt gacgatctca gggtcgtgca ctgactggtt tgaactttca
     2161 gtcttactcc cgttccaggt ggagcggggt tttggagaaa gacgatgagc acaacatccc
     2221 ggccagggct ctgggccctg ataacggccg cggcattcgc gctttgcggc gcgatcctta
     2281 ccgttggcgg cgcatgggtc gctgccatcg gcggccctct ctattatgtc atccttggcc
     2341 tggcacttct cgccacggct ttcctctcct tccggcgcaa tccggctgcc ctctatctgt
     2401 ttgcagtcgt cgtattcgga acggttatct gggaactcac cgttgtcggt ctcgacatct
     2461 gggccctgat cccgcgctcg gacatcgtca tcatcctcgg catctggctg ctgctgccgt
     2521 tcgtctcccg ccagattggc ggcacgcgga cgaccgtcct gcctctcgcc ggcgccgttg
     2581 gcgttgcggt tctggccctg ttcgccagcc tcttcaccga cccgcatgac atcagcggcg
     2641 aactgccgac gcagatcgca aacgcctccc ccgccgaccc ggacaacgtt ccggccagcg
     2701 agtggcacgc ttatggtcgt acgcaggctg gtgaccgctg gtccccgctg aaccagatca
     2761 acgcgtccaa cgtcagcaac ctcaaggtcg catggcatat ccacaccaag gatatgatga
     2821 actccaacga cccgggcgaa gcaacgaacg aagcgacccc gatcgagttc aacaacacgc
     2881 tttacatgtg ctcgctgcac cagaagtcga cctgcaggca tgcaggcttg gcactggccg
     2941 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     3001 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     3061 aacagttgcg cagcctgaat ggcgaatggc gataagctag cttcacgctg ccgcaagcac
     3121 tcagggcgca agggctgcta aaggaagcgg aacacgtaga aagccagtcc gcagaaacgg
     3181 tgctgacccc ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca
     3241 aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta
     3301 tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc
     3361 tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga
     3421 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3481 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3541 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3601 aagaccgacc tgtccggtgc cctgaatgaa ctccaagacg aggcagcgcg gctatcgtgg
     3661 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3721 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3781 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3841 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3901 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3961 ctgttcgcca ggctcaaggc gcggatgccc gacggcgagg atctcgtcgt gacccatggc
     4021 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4081 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4141 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4201 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4261 ggttcggatc cgggcttatc gactgcacgg tgcaccaatg cttctggcgt caggcagcca
     4321 tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag
     4381 gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat
     4441 tcatgacagc ttaagatgcg tgacaaaata cggccgcgct ttcatacatc gtcagaagac
     4501 gcctttcaga ttttccggag aaccaaacag caaaaagggg gaatttcgtg tcgcaagata
     4561 acaactttag ccaggggcca gtcccgcagt cggcgcggaa aggggtattg gcattgacgt
     4621 tcgtcatgct gggattaacc ttcttttccg ccagtatgtg gaccggcggc actctcggaa
     4681 ccggtcttag ctatcatgat ttcttcctcg cagttctcat cggtaatctt ctcctcggta
     4741 tttacacttc atttctcggt tacattggcg caaaaaccgg cctgaccact catcttcttg
     4801 ctcgcttctc gtttggtgtt aaaggctcat ggctgccttc actgctactg ggcggaactc
     4861 aggttggctg gtttggcgtc ggtgtggcga tgtttgccat tccggtgggt aaggcaaccg
     4921 ggctggatat taatttgctg attgccgttt ccggtttact gatgaccgtc accgtctttt
     4981 ttggcatttc ggcgctgacg gttctttcgg tgattgcggt tccggctatc gcctgcctgg
     5041 gcggttattc cgtgtggctg gctgttaacg gcatgggcgg cctggacgca ttaaaagcgg
     5101 tcgttcccgc acaaccgtta gatttcaatg tcgcgctggc gctggttgtg gggtcattta
     5161 tcagtgcggg tacgctcacc gctgactttg tccggtttgg tcgcaatgcc aaactggcgg
     5221 tgctggtggc gatggtggcc tttttcctcg gcaactcgtt gatgtttatt ttcggtgcag
     5281 cgggcgctgc ggcactgggc atggcggata tctctgatgt gatgattgct cagggcctgc
     5341 tgctgcctgc gattgtggtg ctggggctga atatctggac caccaacgat aacgcactct
     5401 atgcgtcggg tttaggtttc gccaacatta ccgggatgtc gagcaaaacc ctttcggtaa
     5461 tcaacggtat tatcggtacg gtctgcgcat tatggctgta taacaatttt gtcggctggt
     5521 tgaccttcct ttcggcagct attcctccag tgggtggcgt gatcatcgcc gactatctga
     5581 tgaaccgtcg ccgctatgag cactttgcga ccacgcgtat gatgagtgtc aattgggtgg
     5641 cgattctggc ggtcgccttg gggattgctg caggccactg gttaccggga attgttccgg
     5701 tcaacgcggt attaggtggc gcgctgagct atctgatcct taacccgatt ttgaatcgta
     5761 aaacgacagc agcaatgacg catgtggagg ctaacagtgt cgaataacgc tttacaaaca
     5821 attattaacg cccggttacc aggcgaagag gggctgtggc agattcatct gcaggacgga
     5881 aaaatcagcg ccattgatgc gcaatccggc gtgatgccca taactgaaaa cagcctggat
     5941 gccgaacaag gtttagttat accgccgttt gtggagccac atattcacct ggacaccacg
     6001 caaaccgccg gacaaccgaa ctggaatcag tccggcacgc tgtttgaagg cattgaacgc
     6061 tgggccgagc gcaaagcgtt attaacccat gacgatgtga aacaacgcgc atggcaaacg
     6121 ctgaaatggc agattgccaa cggcattcag catgtgcgta cccatgtcga tgtttcggat
     6181 gcaacgctaa ctgcgctgaa agcaatgctg gaagtgaagc aggaagtcgc gccgtggatt
     6241 gatctgcaaa tcgtcgcctt ccctcaggaa gggattttgt cgtatcccaa cggtgaagcg
     6301 ttgctggaag aggcgttacg cttaggggca gatgtagtgg gggcgattcc gcattttgaa
     6361 tttacccgtg aatacggcgt ggagtcgctg cataaaacct tcgccctggc gcaaaaatac
     6421 gaccgtctca tcgacgttca ctgtgatgag atcgatgacg agcagtcgcg ctttgtcgaa
     6481 accgttgctg ccctggcgca ccatgaaggc atgggcgcgc gagtcaccgc cagccacacc
     6541 acggcaatgc actcctataa cggggcgtat acctcacgcc tgttccgctt gctgaaaatg
     6601 tccggtatta actttgtcgc caacccgctg gtcaatattc atctgcaagg acgtttcgat
     6661 acgtatccaa aacgtcgcgg catcacgcgc gttaaagaga tgctggagtc cggcattaac
     6721 gtctgctttg gtcacgatga tgtcttcgat ccgtggtatc cgctgggaac ggcgaatatg
     6781 ctgcaagtgc tgcatatggg gctgcatgtt tgccagttga tgggctacgg gcagattaac
     6841 gatggcctga atttaatcac ccaccacagc gcaaggacgt tgaatttgca ggattacggc
     6901 attgccgccg gaaacagcgc caacctgatt atcctgccgg ctgaaaatgg gtttgatgcg
     6961 ctgcgccgtc aggttccggt acgttattcg gtacgtggcg gcaaggtgat tgccagcaca
     7021 caaccggcac aaaccaccgt atatctggag cagccagaag ccatcgatta caaacgttga
     7081 acgactgggt tacagcgagc ttagtttatg ccggatctct cctggcgcac catcgtcggc
     7141 tacagcctcg ggaattgcta ccgcgaaatg accgaccaag cgacgcccaa cctgccatca
     7201 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     7261 gacgccctcg cggacgtgct catagtccac gacgcccgtg attttgtagc cctggccgac
     7321 ggccagcagg taggccgaca ggctcatgcc ggccgccgcc gccttttcct caatcgctct
     7381 tcgttcgtct ggaaggcagt acaccttgat aggtgggctg cccttcctgg ttggcttggt
     7441 ttcatcagcc atccgcttgc cctcatctgt tacgccggcg gtagccggcc agcctcgcag
     7501 agcaggattc ccgttgagca ccgccaggtg cgaataaggg acagtgaaga aggaacaccc
     7561 gctcgcgggt gggcctactt cacctatcct gcccggctga cgccgttgga tacaccaagg
     7621 aaagtctaca cgaacccttt ggcaaaatcc tgtatatcgt gcgaaaaagg atggatatac
     7681 cgaaaaaatc gctataatga ccccgaagca gggttatgca gcggaaaagc gctgcttccc
     7741 tgctgttttg tggaatatct accgactgga aacaggcaaa tgcaggaaat tactgaactg
     7801 aggggacagg cgagagacga tgccaaagag ctcctgaaaa tctcgataac tcaaaaaata
     7861 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa
     7921 cgtctcattt tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat
     7981 ttatttattc tgcgaagtga tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg
     8041 gtgatgctgc caacttactg atttagtgta tgatggtgtt tttgaggtgc tccagtggct
     8101 tctgtttcta tcagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
     8161 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca
     8221 aaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt attctgcgaa
     8281 gtgatcttcc gtcacaggta tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt
     8341 actgatttag tgtatgatgg tgtttttgag gtgctccagt ggcttctgtt tctatcaggg
     8401 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaaaaggat
     8461 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     8521 ccactgagcg tcagaccccg tagaaaagat caaaggatct tc
//
