LOCUS       sequence-371207-        7180 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Non-replicating vector used to create markerless deletion of 
            RR42_RS18960 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 Apr 14, 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: RS18960_pMQ30_KO_Kan
FEATURES             Location/Qualifiers
     source          1..7180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1039..1057
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1095..1117)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1217..1236
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(1275..2609,2610..2696))
                     /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"
     promoter        complement(2697..3142)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     oriT            3516..3625
                     /note="incP origin of transfer"
     terminator      complement(4068..4095)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4187..4273)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4426..4443
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(4429..4707)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA
                     AHPPFASWRNSEEARTDRPSQQLRSLNGEWRAWLFWRMREDFQPDTD"
     primer_bind     4608..4630
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4622..4639
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4623..4639
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    4643..4699
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4708..4724)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4708..4724)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4721..4743)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4732..4748
                     /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)."
     promoter        complement(join(4756..4762,4763..4780,4781..4786))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4801..4822
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5110..5698)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5190..5209)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5951..6745)
                     /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(6062..6081)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6672..6691
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6934..6939,6940..6956,6957..6962))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        complement(6980..7084)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gctcggcccg aacagcggca gcaccaggta cgggccggcc ggcacgcccc atacgcccag
       61 ggtctggccg aagtcttcct tgtacttcgg caagcccgcc ggcgtggcga tgtcgatcag
      121 cccgccgatg cccagcacgc tgttgatggc cacgcgcatg gtgtcttccg cggcgcgggt
      181 gggcttgccc tgcagcaggt tgttgacggc cgagtagaca tcgctgacgt tggaaaaggc
      241 attgtcgatc gcggtgcgga tcggcgacgg aacgtaatcg gcgtacacct gggcaaccgg
      301 gcgcagcacg gtcctgtcga cggcgtcgtt aaccttgaag acttcccggt tgaagggctc
      361 gataggatcc ttcgggttgg cggtcgggcc agtggcgcaa cctgccgcca gccatgcaac
      421 cgaggccgcc agcaagcagt ttcgtcccac gcgccagcgc gcggcagcac gcctgccggt
      481 catttcacca cccccgctgc ctgcgtctct tcttcaagta ctccggctcc ggcgaccgca
      541 ccggccatgt cagctgaaca tcagcgcagt cagcaggaaa tccagcgcca gcaccgccag
      601 cgaggcaatc acgacagtgc gcgtggtggc gcgcgacacg ccttccggcg tcggcttggc
      661 ctgatagccc tggtacaacg ctataaacgt caccgcgacg ccaaaaatga agctcttaat
      721 gacgccattg agcacgtcat tgcgaacatc cacgccgttt tgcatctgcg accagaacgc
      781 gcccgcgtcc acgccgatca tctgcacccc gaccacatag ccacccagga tgccaacggc
      841 gctgaaaatc gccgccagca ccggcatggc aaccacacca gcccagaagc gcggcgcaat
      901 cacgcgctgc agcggattga ccgccatcat ttccatggcg gtgagttgct cacccgcctt
      961 catcaggccg atctcggcgg tgagtgacgt gccggcacgg tctaagaaac cattattatc
     1021 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     1081 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     1141 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     1201 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca taatcggcat
     1261 tttcttttgc gtttttattt gttaactgtt aattgtcctt gttcaaggat gctgtctttg
     1321 acaacagatg ttttcttgcc tttgatgttc agcaggaagc taggcgcaaa cgttgattgt
     1381 ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa tcacgacatt gtttcctttc
     1441 gcttgaggta cagcgaagtg tgagtaagta aaggttacat cgttaggatc aagatccatt
     1501 tttaacacaa ggccagtttt gttcagcggc ttgtatgggc cagttaaaga attagaaaca
     1561 taaccaagca tgtaaatatc gttagacgta atgccgtcaa tcgtcatttt tgatccgcgg
     1621 gagtcagtga acagatacca tttgccgttc attttaaaga cgttcgcgcg ttcaatttca
     1681 tctgttactg tgttagatgc aatcagcggt ttcatcactt ttttcagtgt gtaatcatcg
     1741 tttagctcaa tcataccgag agcgccgttt gctaactcag ccgtgcgttt tttatcgctt
     1801 tgcagaagtt tttgactttc ttgacggaag aatgatgtgc ttttgccata gtatgctttg
     1861 ttaaataaag attcttcgcc ttggtagcca tcttcagttc cagtgtttgc ttcaaatact
     1921 aagtatttgt ggcctttatc ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct
     1981 gagctgtagt tgccttcatc gatgaactgc tgtacatttt gatacgtttt tccgtcaccg
     2041 tcaaagattg atttataatc ctctacaccg ttgatgttca aagagctgtc tgatgctgat
     2101 acgttaactt gtgcagttgt cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg
     2161 tagaataaac ggatttttcc gtcagatgta aatgtggctg aacctgacca ttcttgtgtt
     2221 tggtctttta ggatagaatc atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca
     2281 gcgtttttcc agctgtcaat agaagtttcg ccgacttttt gatagaacat gtaaatcgat
     2341 gtgtcatccg catttttagg atctccggct aatgcaaaga cgatgtggta gccgtgatag
     2401 tttgcgacag tgccgtcagc gttttgtaat ggccagctgt cccaaacgtc caggcctttt
     2461 gcagaagaga tatttttaat tgtggacgaa tcgaactcag gaacttgata tttttcattt
     2521 ttttgctgtt cagggatttg cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt
     2581 tccttatatg gcttttggtt cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc
     2641 agtgcggtag taaaggttaa tactgttgct tgttttgcaa actttttgat gttcatcgtt
     2701 catgtctcct tttttatgta ctgtgttagc ggtctgcttc ttccagccct cctgtttgaa
     2761 gatggcaagt tagttacgca caataaaaaa agacctaaaa tatgtaaggg gtgacgccaa
     2821 agtatacact ttgcccttta cacattttag gtcttgcctg ctttatcagt aacaaacccg
     2881 cgcgatttac ttttcgacct cattctatta gactctcgtt tggattgcaa ctggtctatt
     2941 ttcctctttt gtttgataga aaatcataaa aggatttgca gactacgggc ctaaagaact
     3001 aaaaaatcta tctgtttctt ttcattctct gtatttttta tagtttctgt tgcatgggca
     3061 taaagttgcc tttttaatca caattcagaa aatatcataa tatctcattt cactaaataa
     3121 tagtgaacgg caggtatatg tgatgggtta aaaaggatcg atcctctagc tagagtcgat
     3181 cttcgccagc agggcgagga tcgtggcatc accgaaccgc gccgtgcgcg ggtcgtcggt
     3241 gagccagagt ttcagcaggc cgcccaggcg gcccaggtcg ccattgatgc gggccagctc
     3301 gcggacgtgc tcatagtcca cgacgcccgt gattttgtag ccctggccga cggccagcag
     3361 gtaggccgac aggctcatgc cggccgccgc cgccttttcc tcaatcgctc ttcgttcgtc
     3421 tggaaggcag tacaccttga taggtgggct gcccttcctg gttggcttgg tttcatcagc
     3481 catccgcttg ccctcatctg ttacgccggc ggtagccggc cagcctcgca gagcaggatt
     3541 cccgttgagc accgccaggt gcgaataagg gacagtgaag aaggaacacc cgctcgcggg
     3601 tgggcctact tcacctatcc tgcccggctg acgccgttgg atacaccaag gaaagtctac
     3661 acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat
     3721 cgctataatg accccgaagc agggttatgc agcggaaaag cgctgcttcc ctgctgtttt
     3781 gtggaatatc taccgactgg aaacaggcaa atgcaagaaa ttactgaact gaggggacag
     3841 gcgagaggcg atgccaaaga gctacaccga cgagctggcc gagtgggttg aatcccgcgc
     3901 ggccaagaag cgccggcgtg atgaggctgc ggttgcgttc ctggcggtga gggcggatgt
     3961 cgatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcat
     4021 atttgaatgt atttagaaaa ataaacaaaa gagtttgtag aaacgcaaaa aggccatccg
     4081 tcaggatggc cttctgctta atttgatgcc tggcagttta tggcgggcgt cctgcccgcc
     4141 accctccggg ccgttgcttc gcaacgttca aatccgctcc cggcggattt gtcctactca
     4201 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct
     4261 ttcgttttat ttgatgcctg gcagttccct actctcgcat ggggagaccc cacactacca
     4321 tcggcgctac ggcgtttcac ttctgagttc ggcatggggt caggtgggac caccgcgcta
     4381 ctgccgccag gcaaattctg ttttatcaga ccgcttctgc gttctgattt aatctgtatc
     4441 aggctgaaaa tcttctctca tccgccaaaa cagccaagct cgccattcgc cattcaggct
     4501 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     4561 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     4621 ttgtaaaacg acggccagtg ccaagcttgc atgcctgcag gtcgactcta gaggatcccc
     4681 gggtaccgag ctcgaattcg taatcatgtc atagctgttt cctgtgtgaa attgttatcc
     4741 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4801 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4861 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4921 tgggcgctct tccgcttcct cgctcactga cccgctgcgc tcggtcgttc ggctgcggcg
     4981 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5041 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5101 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5161 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5221 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5281 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5341 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5401 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5461 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5521 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5581 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5641 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5701 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5761 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5821 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacaatc gatgcgaatt
     5881 ggccgcggcg ttgtgacaat ttaccgaaca actccgcggc cgggaagccg atctcggctt
     5941 gaacgaattg tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg
     6001 gagcggcgat accgtaaagc acgaggaagc gatcagccca ttcaccgcca agctcttcag
     6061 caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac
     6121 agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc
     6181 catgggtcac gacgagatcc tcgccgtcgg gcatccgcgc cttgagcctg gcgaacagtt
     6241 cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt
     6301 ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag
     6361 ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatacc ttctcggcag
     6421 gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc
     6481 ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc
     6541 acgatagccg cgctgcctcg tcttggagtt cattcagggc accggacagg tcggtcttga
     6601 caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcggccga
     6661 ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacccg
     6721 cgtgcaatcc atcttgttca atcatcgttg ctgctccata acatcaaaca tcgacccacg
     6781 gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa
     6841 caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac
     6901 cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc
     6961 aattcggttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7021 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7081 cgcgcacatt tccccgaaaa gtgggtaata actgatataa ttaaattgaa gctctaattt
     7141 gtgagtttag tatacatgca tttacttata atacagtttt
//
