LOCUS       sequence-371206-        7180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Non-replicating vector used to create markerless deletion of 
            RR42_RS35075 in C. basilensis 4G11.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7180)
  AUTHORS   Sander K, Abel AJ, Friedline S, Sharpless W, Skerker J, Deutschbauer
            A, Clark DS, Arkin AP
  TITLE     Eliminating genes for a two-component system increases PHB 
            productivity in Cupriavidus basilensis 4G11 under PHB suppressing, 
            nonstress conditions.
  JOURNAL   Biotechnol Bioeng. 2024 Jan;121(1):139-156. doi: 10.1002/bit.28532. 
            Epub 2023 Aug 28.
  PUBMED    37638652
REFERENCE   2  (bases 1 to 7180)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1002/bit.28532";
            journalName: "Biotechnol Bioeng"; date: "2024-01"; volume: "121"; 
            issue: "1"; pages: "139-156"
COMMENT     Sequence Label: RS35075_pMQ30_KO_Kan
FEATURES             Location/Qualifiers
     source          1..7180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(75..92)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      245..331
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      423..450
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     oriT            893..1002
                     /note="incP origin of transfer"
     promoter        1376..1821
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1822..1908,1909..3243)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     primer_bind     complement(3282..3301)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3401..3423
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3461..3479)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4614..4718
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(4736..4741,4742..4758,4759..4764)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             4953..5747
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGRSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5007..5026)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5617..5636
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      6000..6588
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6489..6508
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6876..6897
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6912..6917,6918..6935,6936..6942)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6950..6966
                     /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     6955..6977
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6974..6990
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6974..6990
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6991..7180,1..89)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA
                     AHPPFASWRNSEEARTDRPSQQLRSLNGEWRAWLFWRMREDFQPDTD"
     misc_feature    6999..7055
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(7059..7076)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7059..7075)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7068..7090)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cttcccaaca gttgcgcagc ctgaatggcg aatggcgagc ttggctgttt tggcggatga
       61 gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag
      121 aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg
      181 aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag
      241 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
      301 gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa
      361 gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa
      421 gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt
      481 tctaaataca ttcaaatatg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
      541 tttcacaccg catatcgaca tccgccctca ccgccaggaa cgcaaccgca gcctcatcac
      601 gccggcgctt cttggccgcg cgggattcaa cccactcggc cagctcgtcg gtgtagctct
      661 ttggcatcgc ctctcgcctg tcccctcagt tcagtaattt cttgcatttg cctgtttcca
      721 gtcggtagat attccacaaa acagcaggga agcagcgctt ttccgctgca taaccctgct
      781 tcggggtcat tatagcgatt ttttcggtat atccatcctt tttcgcacga tatacaggat
      841 tttgccaaag ggttcgtgta gactttcctt ggtgtatcca acggcgtcag ccgggcagga
      901 taggtgaagt aggcccaccc gcgagcgggt gttccttctt cactgtccct tattcgcacc
      961 tggcggtgct caacgggaat cctgctctgc gaggctggcc ggctaccgcc ggcgtaacag
     1021 atgagggcaa gcggatggct gatgaaacca agccaaccag gaagggcagc ccacctatca
     1081 aggtgtactg ccttccagac gaacgaagag cgattgagga aaaggcggcg gcggccggca
     1141 tgagcctgtc ggcctacctg ctggccgtcg gccagggcta caaaatcacg ggcgtcgtgg
     1201 actatgagca cgtccgcgag ctggcccgca tcaatggcga cctgggccgc ctgggcggcc
     1261 tgctgaaact ctggctcacc gacgacccgc gcacggcgcg gttcggtgat gccacgatcc
     1321 tcgccctgct ggcgaagatc gactctagct agaggatcga tcctttttaa cccatcacat
     1381 atacctgccg ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga
     1441 ttaaaaaggc aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag
     1501 aaacagatag attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct
     1561 atcaaacaaa agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg
     1621 tcgaaaagta aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa
     1681 agggcaaagt gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc
     1741 gtaactaact tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac
     1801 ataaaaaagg agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta
     1861 acctttacta ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac
     1921 caaaagccat ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa
     1981 atccctgaac agcaaaaaaa tgaaaaatat caagttcctg agttcgattc gtccacaatt
     2041 aaaaatatct cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct
     2101 gacggcactg tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct
     2161 aaaaatgcgg atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt
     2221 gacagctgga aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat
     2281 tctatcctaa aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga
     2341 aaaatccgtt tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca
     2401 actgcacaag ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat
     2461 tataaatcaa tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat
     2521 gaaggcaact acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat
     2581 aaaggccaca aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc
     2641 gaagaatctt tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa
     2701 agtcaaaaac ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc
     2761 ggtatgattg agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca
     2821 tctaacacag taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg
     2881 tatctgttca ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat
     2941 atttacatgc ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa
     3001 actggccttg tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac
     3061 ttcgctgtac ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga
     3121 ggattctacg cagacaaaca atcaacgttt gcgcctagct tcctgctgaa catcaaaggc
     3181 aagaaaacat ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa
     3241 taaaaacgca aaagaaaatg ccgattatgg tgcactctca gtacaatctg ctctgatgcc
     3301 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3361 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3421 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     3481 ttataggtta atgtcatgat aataatggtt tcttagatca ggccctgccc cagtagcaag
     3541 atcgacaatg ccagcaccat catcggcgtg agcgagccga tgatgcccat gtgggtcgcc
     3601 gtggtcaggt aggcggcata gtaggccagg cactgatagg ccaccatgcc gagcaggccc
     3661 agcacggcga tccgccccaa tcggggccgg atcgcgggcc agttgcgcag cacgggacgc
     3721 agtgccagcg gcgtgaagag cacggctgcc agcaaccagc ggtagaagct gatctcggcc
     3781 ggcgcgatgg tgccggcggc cattttgctg acgacggtgt tgcccgacca gatcacgacg
     3841 gccaggagcg gaaaggcgta ttgccacatg agcgctttct ctgatgtgta tggaacgcta
     3901 tcatgagcgc gtctgtgttg atctatatag ctcaatcagg acataccgac cggaattccg
     3961 gactaattcc ggactatttc cggattccgt atcgcaacag acccaacaac catggcttgt
     4021 ttgcgcttac cgcacaccgc tgagcagcgc cgcgatggct aacgggttgg acgggaaata
     4081 cagatgcgtg taggacgccg tcagcgcgcc ttgccggaac accgcctcgc cccgcgaccc
     4141 gtcgcgcggc acagtggcgt ggcagtaggg tgccagcggg gtctgcgcgc tggaataatg
     4201 gaaggtgtgg ccgcgcaagg tgccttcggg caacgccagt tgttgatggc cgagcgcggc
     4261 aaggcggcgt tgcattgtca ccgtgccagg cagcaggccc gccacggcgt gactatcccc
     4321 ggccttgtcg accagcgtct cgaacaacgc catcatgccg ccgcattcgg cgaggatcgg
     4381 caggctgtcg gcatgggctt ggcgcagggc ggccagcatg cctgtgttgg ctgacaggcg
     4441 cgcggcgtgc agctcggggt agccgccggg cagccagagc gcatcgcagt ccggcaaggt
     4501 agcgtcgtcg agcggggaaa actgtattat aagtaaatgc atgtatacta aactcacaaa
     4561 ttagagcttc aatttaatta tatcagttat tacccacttt tcggggaaat gtgcgcggaa
     4621 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4681 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa ccgaattgac
     4741 ataagcctgt tcggttcgta aactgtaatg caagtagcgt atgcgctcac gcaactggtc
     4801 cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca tggcttgtta
     4861 tgactgtttt tttgtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt
     4921 gggtcgatgt ttgatgttat ggagcagcaa cgatgattga acaagatgga ttgcacgcgg
     4981 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     5041 gccgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     5101 agaccgacct gtccggtgcc ctgaatgaac tccaagacga ggcagcgcgg ctatcgtggc
     5161 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     5221 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     5281 ccgagaaggt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     5341 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     5401 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     5461 tgttcgccag gctcaaggcg cggatgcccg acggcgagga tctcgtcgtg acccatggcg
     5521 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     5581 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     5641 aagagcttgg cggtgaatgg gctgatcgct tcctcgtgct ttacggtatc gccgctcccg
     5701 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgacaa ttcgttcaag
     5761 ccgagatcgg cttcccggcc gcggagttgt tcggtaaatt gtcacaacgc cgcggccaat
     5821 tcgcatcgat tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5881 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5941 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6001 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6061 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6121 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6181 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6241 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6301 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6361 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6421 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6481 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6541 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6601 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6661 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6721 cgcagccgaa cgaccgagcg cagcgggtca gtgagcgagg aagcggaaga gcgcccaata
     6781 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     6841 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     6901 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     6961 taacaatttc acacaggaaa cagctatgac atgattacga attcgagctc ggtacccggg
     7021 gatcctctag agtcgacctg caggcatgca agcttggcac tggccgtcgt tttacaacgt
     7081 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     7141 gccagctggc gtaatagcga agaggcccgc accgatcgcc
//
