LOCUS       sequence-483089-        8591 bp    DNA     circular SYN 13-AUG-2025
DEFINITION  Upregulation of mgdh with P114.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8591)
  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 8591)
  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-URP114
FEATURES             Location/Qualifiers
     source          1..8591
                     /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(2962..2979)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2962..2978)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2971..2993)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3483..4277
                     /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(3537..3556)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4147..4166
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             5829..7109
                     /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            7514..7623
                     /note="incP origin of transfer"
     promoter        complement(7858..7948)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        complement(8142..8232)
                     /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 gaacatcgtg gcagatgttg
     1741 agaaatatcc gcagttcctt ccctggtgtg tgaaggcaag catccggacc cagacggaac
     1801 aggagcttgt ggcggatctg acgatcgggt tcggcccgtt ccgcgagacc ttcaccagcc
     1861 gcgtgacgct ggagcggcct tcgcgtatcc gggtgcgcta cgagaaaggg cctttccgtt
     1921 acctgaataa tgtctggacg ttcacgccgg atccgcgggg ctgtctggtc gatttcttcg
     1981 tggatttcga gttccgttcg cgccttctgc agaatgcgat gggtgtcgtg ttcaatgagg
     2041 gcgtgcgcct gatggtctcc gccttcatca agcgggcacg ggacatttac ggcacgcagg
     2101 cgacgaaagc ggtccccccg gcaccgggcc tttcccaaag gacataaaat ttcgtattat
     2161 ttccacaacc attcggtgcg tgggccgtta caggtctcaa gcaccgtcat ggtgacatga
     2221 aaaggattac gtaatgagca caacatcccg gccagggctc tgggccctga taacggccgc
     2281 ggcattcgcg ctttgcggcg cgatccttac cgttggcggc gcatgggtcg ctgccatcgg
     2341 cggccctctc tattatgtca tccttggcct ggcacttctc gccacggctt tcctctcctt
     2401 ccggcgcaat ccggctgccc tctatctgtt tgcagtcgtc gtattcggaa cggttatctg
     2461 ggaactcacc gttgtcggtc tcgacatctg ggccctgatc ccgcgctcgg acatcgtcat
     2521 catcctcggc atctggctgc tgctgccgtt cgtctcccgc cagattggcg gcacgcggac
     2581 gaccgtcctg cctctcgccg gcgccgttgg cgttgcggtt ctggccctgt tcgccagcct
     2641 cttcaccgac ccgcatgaca tcagcggcga actgccgacg cagatcgcaa acgcctcccc
     2701 cgccgacccg gacaacgttc cggccagcga gtggcacgct tatggtcgta cgcaggctgg
     2761 tgaccgctgg tccccgctga accagatcaa cgcgtccaac gtcagcaacc tcaaggtcgc
     2821 atggcatatc cacaccaagg atatgatgaa ctccaacgac ccgggcgaag caacgaacga
     2881 agcgaccccg atcgagttca acaacacgct ttacatgtgc tcgctgcacc agaagtcgac
     2941 ctgcaggcat gcaagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3001 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3061 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     3121 ataagctagc ttcacgctgc cgcaagcact cagggcgcaa gggctgctaa aggaagcgga
     3181 acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg
     3241 ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta
     3301 catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg
     3361 gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc
     3421 caaggatctg atggcgcagg ggatcaagat ctgatcaaga gacaggatga ggatcgtttc
     3481 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     3541 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     3601 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3661 tccaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     3721 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3781 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3841 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3901 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3961 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cggatgcccg
     4021 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     4081 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     4141 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     4201 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     4261 ttgacgagtt cttctgagcg ggactctggg gttcggatcc gggcttatcg actgcacggt
     4321 gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt
     4381 aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc
     4441 gccgacatca taacggttct ggcaaatatt catgacagct taagatgcgt gacaaaatac
     4501 ggccgcgctt tcatacatcg tcagaagacg cctttcagat tttccggaga accaaacagc
     4561 aaaaaggggg aatttcgtgt cgcaagataa caactttagc caggggccag tcccgcagtc
     4621 ggcgcggaaa ggggtattgg cattgacgtt cgtcatgctg ggattaacct tcttttccgc
     4681 cagtatgtgg accggcggca ctctcggaac cggtcttagc tatcatgatt tcttcctcgc
     4741 agttctcatc ggtaatcttc tcctcggtat ttacacttca tttctcggtt acattggcgc
     4801 aaaaaccggc ctgaccactc atcttcttgc tcgcttctcg tttggtgtta aaggctcatg
     4861 gctgccttca ctgctactgg gcggaactca ggttggctgg tttggcgtcg gtgtggcgat
     4921 gtttgccatt ccggtgggta aggcaaccgg gctggatatt aatttgctga ttgccgtttc
     4981 cggtttactg atgaccgtca ccgtcttttt tggcatttcg gcgctgacgg ttctttcggt
     5041 gattgcggtt ccggctatcg cctgcctggg cggttattcc gtgtggctgg ctgttaacgg
     5101 catgggcggc ctggacgcat taaaagcggt cgttcccgca caaccgttag atttcaatgt
     5161 cgcgctggcg ctggttgtgg ggtcatttat cagtgcgggt acgctcaccg ctgactttgt
     5221 ccggtttggt cgcaatgcca aactggcggt gctggtggcg atggtggcct ttttcctcgg
     5281 caactcgttg atgtttattt tcggtgcagc gggcgctgcg gcactgggca tggcggatat
     5341 ctctgatgtg atgattgctc agggcctgct gctgcctgcg attgtggtgc tggggctgaa
     5401 tatctggacc accaacgata acgcactcta tgcgtcgggt ttaggtttcg ccaacattac
     5461 cgggatgtcg agcaaaaccc tttcggtaat caacggtatt atcggtacgg tctgcgcatt
     5521 atggctgtat aacaattttg tcggctggtt gaccttcctt tcggcagcta ttcctccagt
     5581 gggtggcgtg atcatcgccg actatctgat gaaccgtcgc cgctatgagc actttgcgac
     5641 cacgcgtatg atgagtgtca attgggtggc gattctggcg gtcgccttgg ggattgctgc
     5701 aggccactgg ttaccgggaa ttgttccggt caacgcggta ttaggtggcg cgctgagcta
     5761 tctgatcctt aacccgattt tgaatcgtaa aacgacagca gcaatgacgc atgtggaggc
     5821 taacagtgtc gaataacgct ttacaaacaa ttattaacgc ccggttacca ggcgaagagg
     5881 ggctgtggca gattcatctg caggacggaa aaatcagcgc cattgatgcg caatccggcg
     5941 tgatgcccat aactgaaaac agcctggatg ccgaacaagg tttagttata ccgccgtttg
     6001 tggagccaca tattcacctg gacaccacgc aaaccgccgg acaaccgaac tggaatcagt
     6061 ccggcacgct gtttgaaggc attgaacgct gggccgagcg caaagcgtta ttaacccatg
     6121 acgatgtgaa acaacgcgca tggcaaacgc tgaaatggca gattgccaac ggcattcagc
     6181 atgtgcgtac ccatgtcgat gtttcggatg caacgctaac tgcgctgaaa gcaatgctgg
     6241 aagtgaagca ggaagtcgcg ccgtggattg atctgcaaat cgtcgccttc cctcaggaag
     6301 ggattttgtc gtatcccaac ggtgaagcgt tgctggaaga ggcgttacgc ttaggggcag
     6361 atgtagtggg ggcgattccg cattttgaat ttacccgtga atacggcgtg gagtcgctgc
     6421 ataaaacctt cgccctggcg caaaaatacg accgtctcat cgacgttcac tgtgatgaga
     6481 tcgatgacga gcagtcgcgc tttgtcgaaa ccgttgctgc cctggcgcac catgaaggca
     6541 tgggcgcgcg agtcaccgcc agccacacca cggcaatgca ctcctataac ggggcgtata
     6601 cctcacgcct gttccgcttg ctgaaaatgt ccggtattaa ctttgtcgcc aacccgctgg
     6661 tcaatattca tctgcaagga cgtttcgata cgtatccaaa acgtcgcggc atcacgcgcg
     6721 ttaaagagat gctggagtcc ggcattaacg tctgctttgg tcacgatgat gtcttcgatc
     6781 cgtggtatcc gctgggaacg gcgaatatgc tgcaagtgct gcatatgggg ctgcatgttt
     6841 gccagttgat gggctacggg cagattaacg atggcctgaa tttaatcacc caccacagcg
     6901 caaggacgtt gaatttgcag gattacggca ttgccgccgg aaacagcgcc aacctgatta
     6961 tcctgccggc tgaaaatggg tttgatgcgc tgcgccgtca ggttccggta cgttattcgg
     7021 tacgtggcgg caaggtgatt gccagcacac aaccggcaca aaccaccgta tatctggagc
     7081 agccagaagc catcgattac aaacgttgaa cgactgggtt acagcgagct tagtttatgc
     7141 cggatctctc ctggcgcacc atcgtcggct acagcctcgg gaattgctac cgcgaaatga
     7201 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     7261 aaaggttggg cttcggaatc gttttccggg acgccctcgc ggacgtgctc atagtccacg
     7321 acgcccgtga ttttgtagcc ctggccgacg gccagcaggt aggccgacag gctcatgccg
     7381 gccgccgccg ccttttcctc aatcgctctt cgttcgtctg gaaggcagta caccttgata
     7441 ggtgggctgc ccttcctggt tggcttggtt tcatcagcca tccgcttgcc ctcatctgtt
     7501 acgccggcgg tagccggcca gcctcgcaga gcaggattcc cgttgagcac cgccaggtgc
     7561 gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg ggcctacttc acctatcctg
     7621 cccggctgac gccgttggat acaccaagga aagtctacac gaaccctttg gcaaaatcct
     7681 gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg ctataatgac cccgaagcag
     7741 ggttatgcag cggaaaagcg ctgcttccct gctgttttgt ggaatatcta ccgactggaa
     7801 acaggcaaat gcaggaaatt actgaactga ggggacaggc gagagacgat gccaaagagc
     7861 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg
     7921 aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg
     7981 cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac
     8041 aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttactga tttagtgtat
     8101 gatggtgttt ttgaggtgct ccagtggctt ctgtttctat cagctcctga aaatctcgat
     8161 aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt
     8221 acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc ggtatcaaca
     8281 gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat ttattcggcg
     8341 caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt gtttttgagg
     8401 tgctccagtg gcttctgttt ctatcagggc tggatgatcc tccagcgcgg ggatctcatg
     8461 ctggagttct tcgcccaccc caaaaggatc taggtgaaga tcctttttga taatctcatg
     8521 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     8581 aaaggatctt c
//
