LOCUS       sequence-483091-        8085 bp    DNA     circular SYN 13-AUG-2025
DEFINITION  Knockout of pstB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8085)
  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 8085)
  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-KOpstB
FEATURES             Location/Qualifiers
     source          1..8085
                     /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(2456..2473)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2456..2472)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2465..2487)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2977..3771
                     /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(3031..3050)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3641..3660
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             5323..6603
                     /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            7008..7117
                     /note="incP origin of transfer"
     promoter        complement(7352..7442)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        complement(7636..7726)
                     /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 ggatcctcga tgggaccgcc cggctcgtcc tcggggctgg cgaacgcgat cgtgggcagc
     1081 ctgatccaga ccttcatggc gctgctgatg gcaacgccgc ttggtctcgg atgcgggatc
     1141 tatctttcgg aatacgggac agaaaccaat aagttcgcgt cctgcgtccg ttttgtctcc
     1201 gacgtgctga tgtcggtgcc gtccattctg gtgggcctct tcgtctatca ggttctggtg
     1261 gcgccgttcg ggcatttttc cgcgctcgcg ggttctgttg cgctggcgat cctggcggtg
     1321 ccgatcatcg tgcgtacgac cgaagatatg ctgcggcttg tcccgacctc catgcgcgag
     1381 gccggggcgg ctctgggcgc gacgcgctgg cgggtaacgc tttctctgtg cctgcgttct
     1441 gccaagacgg gcgttctgac gggtattctt ctggctctgg cccgggtgag cggtgaaacg
     1501 gcgcccctgc tgtttacgtc gctcggcaac cagaactggt ctttcagcct caaccggccg
     1561 atggcgagcc tgccggtgac gatctaccag tatgccggtg catcctatga agactgggta
     1621 caactggcct gggccggcgc gctgctggtg acgatgggtg tgctggccat caacattgcc
     1681 gtccgcgttt cggcgcgacg cggatgaccc cgggaacaag gcaggggcgc gtcatggctg
     1741 acagatctca catcgtcaaa agctacgagc aggagctggg gcagctccgc tccatgatgg
     1801 cgcagatggg cggcatcgtg gaaaaccagg tggccctcgc gctttccgcc attgcggacc
     1861 gcaacgagga agccgcggat gcggccgcgc gtcaggaccc ggaagtcgac cagctcgaac
     1921 gcgaggtcga aggccttgcg atccgtctgc tggcccttcg cagccccatg gcaggcgacc
     1981 tgcgcgagat cgtggccgcc ctgaagatca ccggcgatct ggaacgtatt ggcgattacg
     2041 cctcgtccat cgcgcggcgt tccctgcgga tccggatgcc ggaaaaccag atttcgctct
     2101 cgggcctgcg cagcatgggg cgtctggtgc aggaaaacct gcgccggtcg atcgatgcga
     2161 tcacgcagca ggattccaac cgcgccatgg aagtctggca ttcggatcgt gccgtggacg
     2221 agtattacac ggcgctgttc cgcgaactcg tgacgtacat gatggaagat gcccgcaata
     2281 tccgcgcctg cacggaactg ctgttcatcg ccaagaacct cgagcggatc ggcgatcatg
     2341 cgaccaacat cgccgaacgc gtcttctatg ccgtgactgg cgagaatctt ccgtctcttc
     2401 gtccgcgcgg agctccggcg tcggcccagt cgacctgcag gcatgcaagc ttggcactgg
     2461 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     2521 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     2581 cccaacagtt gcgcagcctg aatggcgaat ggcgataagc tagcttcacg ctgccgcaag
     2641 cactcagggc gcaagggctg ctaaaggaag cggaacacgt agaaagccag tccgcagaaa
     2701 cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc
     2761 gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt
     2821 ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
     2881 ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca
     2941 agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3001 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3061 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3121 gtcaagaccg acctgtccgg tgccctgaat gaactccaag acgaggcagc gcggctatcg
     3181 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3241 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3301 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3361 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3421 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3481 gaactgttcg ccaggctcaa ggcgcggatg cccgacggcg aggatctcgt cgtgacccat
     3541 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3601 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3661 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3721 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     3781 tggggttcgg atccgggctt atcgactgca cggtgcacca atgcttctgg cgtcaggcag
     3841 ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc
     3901 aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa
     3961 tattcatgac agcttaagat gcgtgacaaa atacggccgc gctttcatac atcgtcagaa
     4021 gacgcctttc agattttccg gagaaccaaa cagcaaaaag ggggaatttc gtgtcgcaag
     4081 ataacaactt tagccagggg ccagtcccgc agtcggcgcg gaaaggggta ttggcattga
     4141 cgttcgtcat gctgggatta accttctttt ccgccagtat gtggaccggc ggcactctcg
     4201 gaaccggtct tagctatcat gatttcttcc tcgcagttct catcggtaat cttctcctcg
     4261 gtatttacac ttcatttctc ggttacattg gcgcaaaaac cggcctgacc actcatcttc
     4321 ttgctcgctt ctcgtttggt gttaaaggct catggctgcc ttcactgcta ctgggcggaa
     4381 ctcaggttgg ctggtttggc gtcggtgtgg cgatgtttgc cattccggtg ggtaaggcaa
     4441 ccgggctgga tattaatttg ctgattgccg tttccggttt actgatgacc gtcaccgtct
     4501 tttttggcat ttcggcgctg acggttcttt cggtgattgc ggttccggct atcgcctgcc
     4561 tgggcggtta ttccgtgtgg ctggctgtta acggcatggg cggcctggac gcattaaaag
     4621 cggtcgttcc cgcacaaccg ttagatttca atgtcgcgct ggcgctggtt gtggggtcat
     4681 ttatcagtgc gggtacgctc accgctgact ttgtccggtt tggtcgcaat gccaaactgg
     4741 cggtgctggt ggcgatggtg gcctttttcc tcggcaactc gttgatgttt attttcggtg
     4801 cagcgggcgc tgcggcactg ggcatggcgg atatctctga tgtgatgatt gctcagggcc
     4861 tgctgctgcc tgcgattgtg gtgctggggc tgaatatctg gaccaccaac gataacgcac
     4921 tctatgcgtc gggtttaggt ttcgccaaca ttaccgggat gtcgagcaaa accctttcgg
     4981 taatcaacgg tattatcggt acggtctgcg cattatggct gtataacaat tttgtcggct
     5041 ggttgacctt cctttcggca gctattcctc cagtgggtgg cgtgatcatc gccgactatc
     5101 tgatgaaccg tcgccgctat gagcactttg cgaccacgcg tatgatgagt gtcaattggg
     5161 tggcgattct ggcggtcgcc ttggggattg ctgcaggcca ctggttaccg ggaattgttc
     5221 cggtcaacgc ggtattaggt ggcgcgctga gctatctgat ccttaacccg attttgaatc
     5281 gtaaaacgac agcagcaatg acgcatgtgg aggctaacag tgtcgaataa cgctttacaa
     5341 acaattatta acgcccggtt accaggcgaa gaggggctgt ggcagattca tctgcaggac
     5401 ggaaaaatca gcgccattga tgcgcaatcc ggcgtgatgc ccataactga aaacagcctg
     5461 gatgccgaac aaggtttagt tataccgccg tttgtggagc cacatattca cctggacacc
     5521 acgcaaaccg ccggacaacc gaactggaat cagtccggca cgctgtttga aggcattgaa
     5581 cgctgggccg agcgcaaagc gttattaacc catgacgatg tgaaacaacg cgcatggcaa
     5641 acgctgaaat ggcagattgc caacggcatt cagcatgtgc gtacccatgt cgatgtttcg
     5701 gatgcaacgc taactgcgct gaaagcaatg ctggaagtga agcaggaagt cgcgccgtgg
     5761 attgatctgc aaatcgtcgc cttccctcag gaagggattt tgtcgtatcc caacggtgaa
     5821 gcgttgctgg aagaggcgtt acgcttaggg gcagatgtag tgggggcgat tccgcatttt
     5881 gaatttaccc gtgaatacgg cgtggagtcg ctgcataaaa ccttcgccct ggcgcaaaaa
     5941 tacgaccgtc tcatcgacgt tcactgtgat gagatcgatg acgagcagtc gcgctttgtc
     6001 gaaaccgttg ctgccctggc gcaccatgaa ggcatgggcg cgcgagtcac cgccagccac
     6061 accacggcaa tgcactccta taacggggcg tatacctcac gcctgttccg cttgctgaaa
     6121 atgtccggta ttaactttgt cgccaacccg ctggtcaata ttcatctgca aggacgtttc
     6181 gatacgtatc caaaacgtcg cggcatcacg cgcgttaaag agatgctgga gtccggcatt
     6241 aacgtctgct ttggtcacga tgatgtcttc gatccgtggt atccgctggg aacggcgaat
     6301 atgctgcaag tgctgcatat ggggctgcat gtttgccagt tgatgggcta cgggcagatt
     6361 aacgatggcc tgaatttaat cacccaccac agcgcaagga cgttgaattt gcaggattac
     6421 ggcattgccg ccggaaacag cgccaacctg attatcctgc cggctgaaaa tgggtttgat
     6481 gcgctgcgcc gtcaggttcc ggtacgttat tcggtacgtg gcggcaaggt gattgccagc
     6541 acacaaccgg cacaaaccac cgtatatctg gagcagccag aagccatcga ttacaaacgt
     6601 tgaacgactg ggttacagcg agcttagttt atgccggatc tctcctggcg caccatcgtc
     6661 ggctacagcc tcgggaattg ctaccgcgaa atgaccgacc aagcgacgcc caacctgcca
     6721 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     6781 cgggacgccc tcgcggacgt gctcatagtc cacgacgccc gtgattttgt agccctggcc
     6841 gacggccagc aggtaggccg acaggctcat gccggccgcc gccgcctttt cctcaatcgc
     6901 tcttcgttcg tctggaaggc agtacacctt gataggtggg ctgcccttcc tggttggctt
     6961 ggtttcatca gccatccgct tgccctcatc tgttacgccg gcggtagccg gccagcctcg
     7021 cagagcagga ttcccgttga gcaccgccag gtgcgaataa gggacagtga agaaggaaca
     7081 cccgctcgcg ggtgggccta cttcacctat cctgcccggc tgacgccgtt ggatacacca
     7141 aggaaagtct acacgaaccc tttggcaaaa tcctgtatat cgtgcgaaaa aggatggata
     7201 taccgaaaaa atcgctataa tgaccccgaa gcagggttat gcagcggaaa agcgctgctt
     7261 ccctgctgtt ttgtggaata tctaccgact ggaaacaggc aaatgcagga aattactgaa
     7321 ctgaggggac aggcgagaga cgatgccaaa gagctcctga aaatctcgat aactcaaaaa
     7381 atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat
     7441 caacgtctca ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag
     7501 gatttattta ttctgcgaag tgatcttccg tcacaggtat ttattcggcg caaagtgcgt
     7561 cgggtgatgc tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg
     7621 gcttctgttt ctatcagctc ctgaaaatct cgataactca aaaaatacgc ccggtagtga
     7681 tcttatttca ttatggtgaa agttggaacc tcttacgtgc cgatcaacgt ctcattttcg
     7741 ccaaaagttg gcccagggct tcccggtatc aacagggaca ccaggattta tttattctgc
     7801 gaagtgatct tccgtcacag gtatttattc ggcgcaaagt gcgtcgggtg atgctgccaa
     7861 cttactgatt tagtgtatga tggtgttttt gaggtgctcc agtggcttct gtttctatca
     7921 gggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaaaag
     7981 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     8041 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
