LOCUS       sequence-483104-        8350 bp    DNA     circular SYN 13-AUG-2025
DEFINITION  Knockout of pstC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8350)
  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 8350)
  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-KOpstC
FEATURES             Location/Qualifiers
     source          1..8350
                     /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(2168..2184)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2721..2737)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3242..4036
                     /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(3296..3315)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3906..3925
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             5588..6868
                     /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            7273..7382
                     /note="incP origin of transfer"
     promoter        complement(7617..7707)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        complement(7901..7991)
                     /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 ggatcctcta gaagcatctc gaacgctgtc tctgaacaga aaaatgcccc gcaggagacc
     1081 tgcggggcgc tggaagtctc agtcgatgcc gggggtgatc agctccggct gcccttgtga
     1141 tcgccgtcgc gcatctggcc cgtcggctcc tggacatggg cctcaaggaa ccagagatgc
     1201 ttgtcgaggc cacgggacac ttccgtaaag acgtcggccg tatcgtcatc gccagcttcg
     1261 tccgtcacct tgatggactc acgcatgttg ttggcaaccg tcgcataacg gtcgatcagc
     1321 gccgccagat ggtcggccac gcgatagatg ttgttcggat aaggcttgca ggcgctgttt
     1381 ttggaaacat cctgcacggt ccccagtgcc gtcccgccaa gctggacagc acgttcggcg
     1441 accgtatcgg agaagccgtc gatgctggag cggaagccgt cgagcatttc atgaacgccg
     1501 atgaactgcg gtcccttgag attccagtgg gcctgcttgg tgatcagcgc cagatcgatc
     1561 aggtcggaaa gacgggcgtt cagcgtctcg atggaaacat ttttggtgtt gtccgccgtg
     1621 ttgttctgcg tcacgtggcg gtcggttctg gaaaccatgg gaatactcct gaacatcgga
     1681 atgcgacccg aagacgggat tcaggtcgtg aaaatcttct cgtgcccctg actgttccgg
     1741 ggccgtcgct catttaaacg tgtcatccgt gccacggttg cgtcgcgaat tcaggttcgg
     1801 gtcggcaatt tcatgatctg ttgccgggtg aggtgttcgg agctgttgcc cgcgccttcc
     1861 attgaccgtt cacggcggtt tgtaacggcc atggcctgtg gggatgacat aaatctttca
     1921 tcccgtgtcg ttcctgttcc ggttgttctt ccatgacagg acggctaatg cactggtgtg
     1981 gtggctgtag ctgccggata acgcccgttt ttcaagtcag agctcctcac gcatgagcga
     2041 aaccgtcgtc cggcagatgg cgttgtggca cctgcggcct gacgcagccg cgaatatgca
     2101 tcgcgggaca ggtccgagac ggcgaaaacc acgctctaga gtcgacctgc aggcatgcaa
     2161 gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     2221 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     2281 ccgatcgccc ttcccaacag ttstcttata cacaaccgac gccgccgacg aatagagagg
     2341 gtcatcactt catcggggcc cgctcatgtt aatcagaagt gctatgactg cggagctgtt
     2401 gcccgcgcct tccattgacc gttcacggcg gtttgtaacg gccatggcct gtggggatga
     2461 cataaatctt tcatcccgtg tcgttcctgt tccggttgtt cttccatgac aggacggcta
     2521 atgcactggt gtggtggctg tagctgccgg ataacgcccg tttttcaagt cagagctcct
     2581 cacgcatgag cgaaaccgtc gtccggcaga tggcgttgtg gcacctgcgg cctgacgcag
     2641 ccgcgaatat gcatcgcggg acaggtccga gacggcgaaa accacgctct agagtcgacc
     2701 tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     2761 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2821 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcga
     2881 taagctagct tcacgctgcc gcaagcactc agggcgcaag ggctgctaaa ggaagcggaa
     2941 cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc
     3001 tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac
     3061 atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg
     3121 ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc
     3181 aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg
     3241 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3301 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3361 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3421 ccaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3481 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3541 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3601 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3661 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3721 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc ggatgcccga
     3781 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3841 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3901 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3961 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4021 tgacgagttc ttctgagcgg gactctgggg ttcggatccg ggcttatcga ctgcacggtg
     4081 caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta
     4141 aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg
     4201 ccgacatcat aacggttctg gcaaatattc atgacagctt aagatgcgtg acaaaatacg
     4261 gccgcgcttt catacatcgt cagaagacgc ctttcagatt ttccggagaa ccaaacagca
     4321 aaaaggggga atttcgtgtc gcaagataac aactttagcc aggggccagt cccgcagtcg
     4381 gcgcggaaag gggtattggc attgacgttc gtcatgctgg gattaacctt cttttccgcc
     4441 agtatgtgga ccggcggcac tctcggaacc ggtcttagct atcatgattt cttcctcgca
     4501 gttctcatcg gtaatcttct cctcggtatt tacacttcat ttctcggtta cattggcgca
     4561 aaaaccggcc tgaccactca tcttcttgct cgcttctcgt ttggtgttaa aggctcatgg
     4621 ctgccttcac tgctactggg cggaactcag gttggctggt ttggcgtcgg tgtggcgatg
     4681 tttgccattc cggtgggtaa ggcaaccggg ctggatatta atttgctgat tgccgtttcc
     4741 ggtttactga tgaccgtcac cgtctttttt ggcatttcgg cgctgacggt tctttcggtg
     4801 attgcggttc cggctatcgc ctgcctgggc ggttattccg tgtggctggc tgttaacggc
     4861 atgggcggcc tggacgcatt aaaagcggtc gttcccgcac aaccgttaga tttcaatgtc
     4921 gcgctggcgc tggttgtggg gtcatttatc agtgcgggta cgctcaccgc tgactttgtc
     4981 cggtttggtc gcaatgccaa actggcggtg ctggtggcga tggtggcctt tttcctcggc
     5041 aactcgttga tgtttatttt cggtgcagcg ggcgctgcgg cactgggcat ggcggatatc
     5101 tctgatgtga tgattgctca gggcctgctg ctgcctgcga ttgtggtgct ggggctgaat
     5161 atctggacca ccaacgataa cgcactctat gcgtcgggtt taggtttcgc caacattacc
     5221 gggatgtcga gcaaaaccct ttcggtaatc aacggtatta tcggtacggt ctgcgcatta
     5281 tggctgtata acaattttgt cggctggttg accttccttt cggcagctat tcctccagtg
     5341 ggtggcgtga tcatcgccga ctatctgatg aaccgtcgcc gctatgagca ctttgcgacc
     5401 acgcgtatga tgagtgtcaa ttgggtggcg attctggcgg tcgccttggg gattgctgca
     5461 ggccactggt taccgggaat tgttccggtc aacgcggtat taggtggcgc gctgagctat
     5521 ctgatcctta acccgatttt gaatcgtaaa acgacagcag caatgacgca tgtggaggct
     5581 aacagtgtcg aataacgctt tacaaacaat tattaacgcc cggttaccag gcgaagaggg
     5641 gctgtggcag attcatctgc aggacggaaa aatcagcgcc attgatgcgc aatccggcgt
     5701 gatgcccata actgaaaaca gcctggatgc cgaacaaggt ttagttatac cgccgtttgt
     5761 ggagccacat attcacctgg acaccacgca aaccgccgga caaccgaact ggaatcagtc
     5821 cggcacgctg tttgaaggca ttgaacgctg ggccgagcgc aaagcgttat taacccatga
     5881 cgatgtgaaa caacgcgcat ggcaaacgct gaaatggcag attgccaacg gcattcagca
     5941 tgtgcgtacc catgtcgatg tttcggatgc aacgctaact gcgctgaaag caatgctgga
     6001 agtgaagcag gaagtcgcgc cgtggattga tctgcaaatc gtcgccttcc ctcaggaagg
     6061 gattttgtcg tatcccaacg gtgaagcgtt gctggaagag gcgttacgct taggggcaga
     6121 tgtagtgggg gcgattccgc attttgaatt tacccgtgaa tacggcgtgg agtcgctgca
     6181 taaaaccttc gccctggcgc aaaaatacga ccgtctcatc gacgttcact gtgatgagat
     6241 cgatgacgag cagtcgcgct ttgtcgaaac cgttgctgcc ctggcgcacc atgaaggcat
     6301 gggcgcgcga gtcaccgcca gccacaccac ggcaatgcac tcctataacg gggcgtatac
     6361 ctcacgcctg ttccgcttgc tgaaaatgtc cggtattaac tttgtcgcca acccgctggt
     6421 caatattcat ctgcaaggac gtttcgatac gtatccaaaa cgtcgcggca tcacgcgcgt
     6481 taaagagatg ctggagtccg gcattaacgt ctgctttggt cacgatgatg tcttcgatcc
     6541 gtggtatccg ctgggaacgg cgaatatgct gcaagtgctg catatggggc tgcatgtttg
     6601 ccagttgatg ggctacgggc agattaacga tggcctgaat ttaatcaccc accacagcgc
     6661 aaggacgttg aatttgcagg attacggcat tgccgccgga aacagcgcca acctgattat
     6721 cctgccggct gaaaatgggt ttgatgcgct gcgccgtcag gttccggtac gttattcggt
     6781 acgtggcggc aaggtgattg ccagcacaca accggcacaa accaccgtat atctggagca
     6841 gccagaagcc atcgattaca aacgttgaac gactgggtta cagcgagctt agtttatgcc
     6901 ggatctctcc tggcgcacca tcgtcggcta cagcctcggg aattgctacc gcgaaatgac
     6961 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     7021 aaggttgggc ttcggaatcg ttttccggga cgccctcgcg gacgtgctca tagtccacga
     7081 cgcccgtgat tttgtagccc tggccgacgg ccagcaggta ggccgacagg ctcatgccgg
     7141 ccgccgccgc cttttcctca atcgctcttc gttcgtctgg aaggcagtac accttgatag
     7201 gtgggctgcc cttcctggtt ggcttggttt catcagccat ccgcttgccc tcatctgtta
     7261 cgccggcggt agccggccag cctcgcagag caggattccc gttgagcacc gccaggtgcg
     7321 aataagggac agtgaagaag gaacacccgc tcgcgggtgg gcctacttca cctatcctgc
     7381 ccggctgacg ccgttggata caccaaggaa agtctacacg aaccctttgg caaaatcctg
     7441 tatatcgtgc gaaaaaggat ggatataccg aaaaaatcgc tataatgacc ccgaagcagg
     7501 gttatgcagc ggaaaagcgc tgcttccctg ctgttttgtg gaatatctac cgactggaaa
     7561 caggcaaatg caggaaatta ctgaactgag gggacaggcg agagacgatg ccaaagagct
     7621 cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga
     7681 aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt ggcccagggc
     7741 ttcccggtat caacagggac accaggattt atttattctg cgaagtgatc ttccgtcaca
     7801 ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca acttactgat ttagtgtatg
     7861 atggtgtttt tgaggtgctc cagtggcttc tgtttctatc agctcctgaa aatctcgata
     7921 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
     7981 cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag
     8041 ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc
     8101 aaagtgcgtc gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt
     8161 gctccagtgg cttctgtttc tatcagggct ggatgatcct ccagcgcggg gatctcatgc
     8221 tggagttctt cgcccacccc aaaaggatct aggtgaagat cctttttgat aatctcatga
     8281 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     8341 aaggatcttc
//
