LOCUS       sequence-475903-        8610 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Upregulation of mgdh with P112.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8610)
  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 8610)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 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-URP112
FEATURES             Location/Qualifiers
     source          1..8610
                     /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(2981..2998)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2981..2997)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2990..3012)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3502..4296
                     /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(3556..3575)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4166..4185
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             5848..7128
                     /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            7533..7642
                     /note="incP origin of transfer"
     promoter        complement(7877..7967)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        complement(8161..8251)
                     /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 gaacatccag ttccagcagg
     1741 gaacggtcct caaagcctgg ctgttcacaa tacagagaaa tgccttctac gagcagacgc
     1801 gccgtcgcag ccgcgagcag gaaatcatgc tcgacatcga gcaggacatg cagccttcca
     1861 cggctgcacc gtcacgccag aatgcccgcg atgccgttca ggacctggga gaactgctgt
     1921 ggaagctgcc cgatctgttg cgggaggccc tgattctcgt gggagcacag gaactgtcct
     1981 atgaagatgc cgcccgcgtc tgcgatgtgc ccgccggaac gatgagggcc cgtgtttccc
     2041 gtgcccgagc ccagctcgcc gagcttgcga cgaaaaatac cgaacctccg gaaacggaat
     2101 ccgaaaaaaa tatcacctga tttttgcctg atttccgcgg gctccagcct gtcttttcct
     2161 tttttcggga aatttcctgc accccgatgc aacattttcg cctgaccgtt cattgatccc
     2221 atgaaaacag aacgaaatca ggagtcagtt ccatgagcac aacatcccgg ccagggctct
     2281 gggccctgat aacggccgcg gcattcgcgc tttgcggcgc gatccttacc gttggcggcg
     2341 catgggtcgc tgccatcggc ggccctctct attatgtcat ccttggcctg gcacttctcg
     2401 ccacggcttt cctctccttc cggcgcaatc cggctgccct ctatctgttt gcagtcgtcg
     2461 tattcggaac ggttatctgg gaactcaccg ttgtcggtct cgacatctgg gccctgatcc
     2521 cgcgctcgga catcgtcatc atcctcggca tctggctgct gctgccgttc gtctcccgcc
     2581 agattggcgg cacgcggacg accgtcctgc ctctcgccgg cgccgttggc gttgcggttc
     2641 tggccctgtt cgccagcctc ttcaccgacc cgcatgacat cagcggcgaa ctgccgacgc
     2701 agatcgcaaa cgcctccccc gccgacccgg acaacgttcc ggccagcgag tggcacgctt
     2761 atggtcgtac gcaggctggt gaccgctggt ccccgctgaa ccagatcaac gcgtccaacg
     2821 tcagcaacct caaggtcgca tggcatatcc acaccaagga tatgatgaac tccaacgacc
     2881 cgggcgaagc aacgaacgaa gcgaccccga tcgagttcaa caacacgctt tacatgtgct
     2941 cgctgcacca gaagtcgacc tgcaggcatg caagcttggc actggccgtc gttttacaac
     3001 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3061 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3121 gcctgaatgg cgaatggcga taagctagct tcacgctgcc gcaagcactc agggcgcaag
     3181 ggctgctaaa ggaagcggaa cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg
     3241 atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag
     3301 gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc
     3361 gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac
     3421 tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag
     3481 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3541 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3601 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3661 tccggtgccc tgaatgaact ccaagacgag gcagcgcggc tatcgtggct ggccacgacg
     3721 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3781 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3841 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3901 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3961 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4021 ctcaaggcgc ggatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4081 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4141 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4201 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4261 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcggatccg
     4321 ggcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
     4381 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
     4441 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc atgacagctt
     4501 aagatgcgtg acaaaatacg gccgcgcttt catacatcgt cagaagacgc ctttcagatt
     4561 ttccggagaa ccaaacagca aaaaggggga atttcgtgtc gcaagataac aactttagcc
     4621 aggggccagt cccgcagtcg gcgcggaaag gggtattggc attgacgttc gtcatgctgg
     4681 gattaacctt cttttccgcc agtatgtgga ccggcggcac tctcggaacc ggtcttagct
     4741 atcatgattt cttcctcgca gttctcatcg gtaatcttct cctcggtatt tacacttcat
     4801 ttctcggtta cattggcgca aaaaccggcc tgaccactca tcttcttgct cgcttctcgt
     4861 ttggtgttaa aggctcatgg ctgccttcac tgctactggg cggaactcag gttggctggt
     4921 ttggcgtcgg tgtggcgatg tttgccattc cggtgggtaa ggcaaccggg ctggatatta
     4981 atttgctgat tgccgtttcc ggtttactga tgaccgtcac cgtctttttt ggcatttcgg
     5041 cgctgacggt tctttcggtg attgcggttc cggctatcgc ctgcctgggc ggttattccg
     5101 tgtggctggc tgttaacggc atgggcggcc tggacgcatt aaaagcggtc gttcccgcac
     5161 aaccgttaga tttcaatgtc gcgctggcgc tggttgtggg gtcatttatc agtgcgggta
     5221 cgctcaccgc tgactttgtc cggtttggtc gcaatgccaa actggcggtg ctggtggcga
     5281 tggtggcctt tttcctcggc aactcgttga tgtttatttt cggtgcagcg ggcgctgcgg
     5341 cactgggcat ggcggatatc tctgatgtga tgattgctca gggcctgctg ctgcctgcga
     5401 ttgtggtgct ggggctgaat atctggacca ccaacgataa cgcactctat gcgtcgggtt
     5461 taggtttcgc caacattacc gggatgtcga gcaaaaccct ttcggtaatc aacggtatta
     5521 tcggtacggt ctgcgcatta tggctgtata acaattttgt cggctggttg accttccttt
     5581 cggcagctat tcctccagtg ggtggcgtga tcatcgccga ctatctgatg aaccgtcgcc
     5641 gctatgagca ctttgcgacc acgcgtatga tgagtgtcaa ttgggtggcg attctggcgg
     5701 tcgccttggg gattgctgca ggccactggt taccgggaat tgttccggtc aacgcggtat
     5761 taggtggcgc gctgagctat ctgatcctta acccgatttt gaatcgtaaa acgacagcag
     5821 caatgacgca tgtggaggct aacagtgtcg aataacgctt tacaaacaat tattaacgcc
     5881 cggttaccag gcgaagaggg gctgtggcag attcatctgc aggacggaaa aatcagcgcc
     5941 attgatgcgc aatccggcgt gatgcccata actgaaaaca gcctggatgc cgaacaaggt
     6001 ttagttatac cgccgtttgt ggagccacat attcacctgg acaccacgca aaccgccgga
     6061 caaccgaact ggaatcagtc cggcacgctg tttgaaggca ttgaacgctg ggccgagcgc
     6121 aaagcgttat taacccatga cgatgtgaaa caacgcgcat ggcaaacgct gaaatggcag
     6181 attgccaacg gcattcagca tgtgcgtacc catgtcgatg tttcggatgc aacgctaact
     6241 gcgctgaaag caatgctgga agtgaagcag gaagtcgcgc cgtggattga tctgcaaatc
     6301 gtcgccttcc ctcaggaagg gattttgtcg tatcccaacg gtgaagcgtt gctggaagag
     6361 gcgttacgct taggggcaga tgtagtgggg gcgattccgc attttgaatt tacccgtgaa
     6421 tacggcgtgg agtcgctgca taaaaccttc gccctggcgc aaaaatacga ccgtctcatc
     6481 gacgttcact gtgatgagat cgatgacgag cagtcgcgct ttgtcgaaac cgttgctgcc
     6541 ctggcgcacc atgaaggcat gggcgcgcga gtcaccgcca gccacaccac ggcaatgcac
     6601 tcctataacg gggcgtatac ctcacgcctg ttccgcttgc tgaaaatgtc cggtattaac
     6661 tttgtcgcca acccgctggt caatattcat ctgcaaggac gtttcgatac gtatccaaaa
     6721 cgtcgcggca tcacgcgcgt taaagagatg ctggagtccg gcattaacgt ctgctttggt
     6781 cacgatgatg tcttcgatcc gtggtatccg ctgggaacgg cgaatatgct gcaagtgctg
     6841 catatggggc tgcatgtttg ccagttgatg ggctacgggc agattaacga tggcctgaat
     6901 ttaatcaccc accacagcgc aaggacgttg aatttgcagg attacggcat tgccgccgga
     6961 aacagcgcca acctgattat cctgccggct gaaaatgggt ttgatgcgct gcgccgtcag
     7021 gttccggtac gttattcggt acgtggcggc aaggtgattg ccagcacaca accggcacaa
     7081 accaccgtat atctggagca gccagaagcc atcgattaca aacgttgaac gactgggtta
     7141 cagcgagctt agtttatgcc ggatctctcc tggcgcacca tcgtcggcta cagcctcggg
     7201 aattgctacc gcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     7261 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccctcgcg
     7321 gacgtgctca tagtccacga cgcccgtgat tttgtagccc tggccgacgg ccagcaggta
     7381 ggccgacagg ctcatgccgg ccgccgccgc cttttcctca atcgctcttc gttcgtctgg
     7441 aaggcagtac accttgatag gtgggctgcc cttcctggtt ggcttggttt catcagccat
     7501 ccgcttgccc tcatctgtta cgccggcggt agccggccag cctcgcagag caggattccc
     7561 gttgagcacc gccaggtgcg aataagggac agtgaagaag gaacacccgc tcgcgggtgg
     7621 gcctacttca cctatcctgc ccggctgacg ccgttggata caccaaggaa agtctacacg
     7681 aaccctttgg caaaatcctg tatatcgtgc gaaaaaggat ggatataccg aaaaaatcgc
     7741 tataatgacc ccgaagcagg gttatgcagc ggaaaagcgc tgcttccctg ctgttttgtg
     7801 gaatatctac cgactggaaa caggcaaatg caggaaatta ctgaactgag gggacaggcg
     7861 agagacgatg ccaaagagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg
     7921 atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaacg tctcattttc
     7981 gccaaaagtt ggcccagggc ttcccggtat caacagggac accaggattt atttattctg
     8041 cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca
     8101 acttactgat ttagtgtatg atggtgtttt tgaggtgctc cagtggcttc tgtttctatc
     8161 agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg
     8221 gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggccca
     8281 gggcttcccg gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt
     8341 cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct gccaacttac tgatttagtg
     8401 tatgatggtg tttttgaggt gctccagtgg cttctgtttc tatcagggct ggatgatcct
     8461 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aaaaggatct aggtgaagat
     8521 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     8581 agaccccgta gaaaagatca aaggatcttc
//
