LOCUS       sequence-476311-        7928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Electroporation vector for expression of the lambda Red 
            recombineering system in Cupriavidus necator..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7928)
  AUTHORS   Arhar S, Pirchner J, Stolterfoht-Stock H, Reicher K, Kourist R, 
            Emmerstorfer-Augustin A
  TITLE     CnRed: Efficient, Marker-free Genome Engineering of Cupriavidus 
            necator H16 by Adapted Lambda Red Recombineering.
  JOURNAL   ACS Synth Biol. 2025 Feb 24. doi: 10.1021/acssynbio.4c00757.
  PUBMED    39989320
REFERENCE   2  (bases 1 to 7928)
  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"; journalName: "ACS Synth 
            Biol. 2025 Feb 24. doi: 10.1021/acssynbio.4c00757."
COMMENT     Sequence Label: pCnRed
FEATURES             Location/Qualifiers
     source          1..7928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(184..203)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      1247..1682
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     CDS             complement(1719..2690)
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein of plasmid pSa"
                     /label=RepA
                     /translation="MPKNNKAPGHRINEIIKTSLALEMEDAREAGLVGYMARCLVQATM
                     PHTDPKTSNFERTNGIVTLSIMGKPSIGLPYGSMPRTLLAWICTEAVRTKDPVLNLGRS
                     QSEFLQRLGMHTDGRYTATLRNQAQRLFSSMISLAGEQGNDFGIENVVIAKRAFLFWNP
                     KRPEDRALWDSTLTLTGDFFEEVTRSPVPIRIDYLHALRQSPLAMDIYTWLTYRVFLLR
                     AKGRPFVQIPWVALQAQFGSSYGSRARNSPELDDKARERAERAALASFKYNFKKRLREV
                     LIVYPEASDCIEDDGECLRIKSTRLHVTRAPGKGARIGPPPT"
     terminator      complement(3537..3564)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3588..4436)
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(4510..5346)
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     promoter        5492..5610
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             5641..6426
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     CDS             6441..7121
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /label=Exo
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     CDS             7136..7552
                     /codon_start=1
                     /gene="gam"
                     /product="inhibitor of the host RecBCD nuclease in the 
                     lambda Red system"
                     /label=Gam
                     /note="protects the double-stranded DNA substrate for 
                     recombination"
                     /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
                     IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
                     AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
     terminator      7553..7580
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     7639..7657
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(7684..7689,7690..7706,7707..7712)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             join(7760..7928,1..1022)
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
ORIGIN
        1 ttctatgcgc acccgttctc ggagcactgt ccgaccgctt tggccgccgc ccagtcctgc
       61 tcgcttcgct acttggagcc actatcgact acgcgatcat ggcgaccaca cccgtcctgt
      121 ggatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg
      181 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
      241 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
      301 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
      361 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg accgatgccc ttgagagcct
      421 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
      481 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg
      541 gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa
      601 tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga
      661 agcaggccat tatcgccggc atggcggccg acgcgctggg ctacgtcttg ctggcgttcg
      721 cgacgcgagg ctggatggcc ttccccatta tgattcttct cgcttccggc ggcatcggga
      781 tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga cgaccatcag ggacagcttc
      841 aaggatcgct cgcggctctt accagcctaa cttcgatcat tggaccgctg atcgtcacgg
      901 cgatttatgc cgcctcggcg agcacatgga acgggttggc atggattgta ggcgccgccc
      961 tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct
     1021 gaatggaagc cggcggcacc tcgctaacgg attcaccact ccaatttaaa tatcacataa
     1081 attaattaaa gatctcaaac tgtacgttta aacgctactt tccgaacgac tcctgcgttg
     1141 atcggaaatc cagaagcccg agaggttgcc gcctttcggg ctttttcttt ttcaaaaaaa
     1201 aaaatttata aaacgatctg ttgcggccgc cgggttgtgg gcaaaggcgc tcgacggtgg
     1261 gcaaccgctt gcggttgtcc acgggcggag ccggtgcgcg tagcgcattg tccacaagcc
     1321 aagggcgacc aataattgat atatatattc ataattgaaa agctaattga acatactact
     1381 tgctgtaact acttgccgga gcgaggggtg tttgcaagct gttgatctga aagggctatt
     1441 agcgttctca cgtgcctttt tgattagcga tttcacgtga ccttattagc gatttcacgt
     1501 actccgatta gcgatttcac gtaccctgat tagcgatttc acgtggatag tttttggagc
     1561 gggccggaaa gccccgtgaa tcaaggcttt gcggggcatt agcggtttca cgtggataac
     1621 taccctctat ccacaggctt ccggggataa aaaagcccgc tcgacggcgg gctgttggat
     1681 gggaaggctt gaccaagcca agcgtagcgt tggcctggtc aagtcggagg ggggccgatg
     1741 cgagcgccct tgccgggtgc gcgggtgaca tgcaggcgtg tggatttgat gcgcaggcat
     1801 tcgccgtcat cttcgatgca gtcgcttgcc tcgggataga caatcaacac ttcgcgtagg
     1861 cgctttttga agttgtattt gaagctggcg agtgctgccc gctctgcccg ctctcgggcc
     1921 ttatcgtcca gttcgggcga gttgcgtgcg cggctgccat aggatgagcc gaattgcgct
     1981 tgcagggcga cccaagggat ttgcacgaag gggcggccct tggcccgcaa caggaacacg
     2041 cgataggtca gccacgtgta aatgtccatc gcaagcggag actgccgcaa ggcatgcagg
     2101 tagtcgattc ggataggaac cggtgagcgg gtgacttcct cgaagaaatc gcctgtgagg
     2161 gtgagggtgc tatcccatag cgcccgatct tctggccgct tgggattcca gaatagaaaa
     2221 gcgcgcttgg caatgacgac gttctcaatg ccgaagtcat tgccttgctc gccggcaagc
     2281 gaaatcatgg atgaaaacag gcgttgcgcc tgattgcgaa gggtggccgt gtaacggcca
     2341 tcggtgtgca ttccgagcct ttgtagaaat tccgattgcg accggccaag gttcaacacg
     2401 gggtctttcg ttcgcacggc ctcggtgcat atccaagcaa gcaaggtgcg cggcatagaa
     2461 ccgtagggca ggccgatgct cggcttgccc atgatcgaca aggtgacgat gccattggtg
     2521 cgctcaaagt tgctggtctt ggggtcggtg tggggcatgg tcgcttgcac aaggcaacgg
     2581 gccatgtagc cgactaagcc agcttcgcgg gcatcctcca tttcgagcgc gaggctcgtc
     2641 ttgatgatct cgttgatacg atggccgggg gctttgttgt tcttaggcat gttgttccct
     2701 ccccggcatg gtgatggttg gtctagtgtt tgtgggtttg atgttccggc gtttgatgaa
     2761 caggcgcaag gtgtgagggc tgacgcctaa caactcggct gcgcgacttt gcggcaagcc
     2821 aaggttcacg tatgcctgta cttcatcaat acggctgtcc agcttcaagg cgctcgattt
     2881 gctgcccttg ggtcgcccga gcgtcttgcc gcgctctctg gcgacttgta gcgcctcggt
     2941 ggtacgtgcc tgaatgaaat gccgctcgat ctgtgcagcc aagccaagca cggttgccat
     3001 gatgtcgctt tgtaggctgc cgtccatgat gatcttctgt ttggtcacat ggacgattag
     3061 gccgcgctcg ctcgccgctt tgagaatttc caaggcggcg agggcggaac cggcaatgcg
     3121 cgtaatctcc ggcgtcagta gcacgtcgcc acgctcggcc ttttcgatga ttgctccgag
     3181 cttgcgcttg cgccagtcct ttgctctgct ggcaatttct tcctcgatct gtagcggcgc
     3241 gaagcctttg gcgttcgcgt attcgagcaa accgtatttt tggttttccg ggtcttggcc
     3301 gtcacgcgaa acccggagat aggcatagta ttttggcatt tgcagggaaa acgtcagatt
     3361 cggttaaaca tgcctcattc tagcgcagat taaataggaa ttaaataccc tgttgcggta
     3421 tagataaaac gttggtttgt tctgccctat gagcgtacaa aaaaggccgg gtgagtggcc
     3481 cggccttcgt ttaggtgctg aataggattg gttctggtct gccctgcagg tctagacgca
     3541 gaaaggccca cccgaaggtg agccagtgtg actctagtag tttaaactta atctttctgc
     3601 gaattgagat gacgccactg gctgggcgtc atcccggttt cccgggtaaa caccaccgaa
     3661 aaatagttac tatcttcaaa gccacattcg gtcgaaatat cactgattaa caggcggcta
     3721 tgctggagaa gatattgcgc atgacacact ctgacctgtc gcagatattg attgatggtc
     3781 attccagtct gctggcgaaa ttgctgacgc aaaacgcgct cactgcacga tgcctcatca
     3841 caaaatttat ccagcgcaaa gggacttttc aggctagccg ccagccgggt aatcagctta
     3901 tccagcaacg tttcgctgga tgttggcggc aacgaatcac tggtgtaacg atggcgattc
     3961 agcaacatca ccaactgccc gaacagcaac tcagccattt cgttagcaaa cggcacatgc
     4021 tgactacttt catgctcaag ctgaccgata acctgccgcg cctgcgccat ccccatgcta
     4081 cctaagcgcc agtgtggttg ccctgcgctg gcgttaaatc ccggaatcgc cccctgccag
     4141 tcaagattca gcttcagacg ctccgggcaa taaataatat tctgcaaaac cagatcgtta
     4201 acggaagcgt aggagtgttt atcgtcagca tgaatgtaaa agagatcgcc acgggtaatg
     4261 cgataagggc gatcgttgag tacatgcagg ccattaccgc gccagacaat caccagctca
     4321 caaaaatcat gtgtatgttc agcaaagaca tcttgcggat aacggtcagc cacagcgact
     4381 gcctgctggt cgctggcaaa aaaatcatct ttgagaagtt ttaactgatg cgccaccgtg
     4441 gctacctcgg ccagagaacg aagttgatta ttcgcaatat ggcgtacaaa tacgttgaga
     4501 agattcgcgt tattgcagaa agccatcccg tccctggcga atatcacgcg gtgaccagtt
     4561 aaactctcgg cgaaaaagcg tcgaaaagtg gttactgtcg ctgaatccac agcgataggc
     4621 gatgtcagta acgctggcct cgctgtggcg tagcagatgt cgggctttca tcagtcgcag
     4681 gcggttcagg tatcgctgag gcgtcagtcc cgtttgctgc ttaagctgcc gatgtagcgt
     4741 acgcagtgaa agagaaaatt gatccgccac ggcatcccaa ttcacctcat cggcaaaatg
     4801 gtcctccagc caggccagaa gcaagttgag acgtgatgcg ctgttttcca ggttctcctg
     4861 caaactgctt ttacgcagca agagcagtaa ttgcataaac aagatctcgc gactggcggt
     4921 cgagggtaaa tcattttccc cttcctgctg ttccatctgt gcaaccagct gtcgcacctg
     4981 ctgcaatacg ctgtggttaa cgcgccagtg agacggatac tgcccatcca gctcttgtgg
     5041 cagcaactga ttcagcccgg cgagaaactg aaatcgatcc ggcgagcgat acagcacatt
     5101 ggtcagacac agattatcgg tatgttcata cagatgccga tcatgatcgc gtacgaaaca
     5161 gaccgtgcca ccggtgatgg tatagggctg cccattaaac acatgaatac ccgtgccatg
     5221 ttcgacaatc acaatttcat gaaaatcatg atgatgttca ggaaaatccg cctgcgggag
     5281 ccggggttct atcgccacgg acgcgttacc agacggaaaa aaatccacac tatgtaatac
     5341 ggtcatactg gcctcctgat gtcgtcaaca cggcgaaata gtaatcacga ggtcaggttc
     5401 ttaccttaaa ttttcgacgg aaaaccacgt aaaaaacgtc gatttttcaa gatacagcgt
     5461 gaattttcag gaaatgcggt gagcatcaca tcaccacaat tcagcaaatt gtgaacatca
     5521 tcacgttcat ctttccctgg ttgccaatgg cccattttcc tgtcagtaac gagaaggtcg
     5581 cgaattcagg cgctttttag actggtcgta atgaacaatt cttaagaagg agatatacat
     5641 atgagtactg cactcgcaac gctggctggg aagctggctg aacgtgtcgg catggattct
     5701 gtcgacccac aggaactgat caccactctt cgccagacgg catttaaagg tgatgccagc
     5761 gatgcgcagt tcatcgcatt actgatcgtt gccaaccagt acggccttaa tccgtggacg
     5821 aaagaaattt acgcctttcc tgataagcag aatggcatcg ttccggtggt gggcgttgat
     5881 ggctggtccc gcatcatcaa tgaaaaccag cagtttgatg gcatggactt tgagcaggac
     5941 aatgaatcct gtacatgccg gatttaccgc aaggaccgta atcatccgat ctgcgttacc
     6001 gaatggatgg atgaatgccg ccgcgaacca ttcaaaactc gcgaaggcag agaaatcacg
     6061 gggccgtggc agtcgcatcc caaacggatg ttacgtcata aagccatgat tcagtgtgcc
     6121 cgtctggcct tcggatttgc tggtatctat gacaaggatg aagccgagcg cattgtcgaa
     6181 aatactgcat acactgcaga acgtcagccg gaacgcgaca tcactccggt taacgatgaa
     6241 accatgcagg agattaacac tctgctgatc gccctggata aaacatggga tgacgactta
     6301 ttgccgctct gttcccagat atttcgccgc gacattcgtg catcgtcaga actgacacag
     6361 gccgaagcag taaaagctct tggattcctg aaacagaaag ccgcagagca gaaggtggca
     6421 gcatgaaaag gaggacaacc atgacaccgg acattatcct gcagcgtacc gggatcgatg
     6481 tgagagctgt cgaacagggg gatgatgcgt ggcacaaatt acggctcggc gtcatcaccg
     6541 cttcagaagt tcacaacgtg atagcaaaac cccgctccgg aaagaagtgg cctgacatga
     6601 aaatgtccta cttccacacc ctgcttgctg aggtttgcac cggtgtggct ccggaagtta
     6661 acgctaaagc actggcctgg ggaaaacagt acgagaacga cgccagaacc ctgtttgaat
     6721 tcacttccgg cgtgaatgtt actgaatccc cgatcatcta tcgcgacgaa agtatgcgta
     6781 ccgcctgctc tcccgatggt ttatgcagtg acggcaacgg ccttgaactg aaatgcccgt
     6841 ttacctcccg ggatttcatg aagttccggc tcggtggttt cgaggccata aagtcagctt
     6901 acatggccca ggtgcagtac agcatgtggg tgacgcgaaa aaatgcctgg tactttgcca
     6961 actatgaccc gcgtatgaag cgtgaaggcc tgcattatgt cgtgattgag cgggatgaaa
     7021 agtacatggc gagttttgac gagatcgtgc cggagttcat cgaaaaaatg gacgaggcac
     7081 tggctgaaat tggttttgta tttggggagc aatggcgatg aaaaggagga caaccatgga
     7141 tattaatact gaaactgaga tcaagcaaaa gcattcacta accccctttc ctgttttcct
     7201 aatcagcccg gcatttcgcg ggcgatattt tcacagctat ttcaggagtt cagccatgaa
     7261 cgcttattac attcaggatc gtcttgaggc tcagagctgg gcgcgtcact accagcagct
     7321 cgcccgtgaa gagaaagagg cagaactggc agacgacatg gaaaaaggcc tgccccagca
     7381 cctgtttgaa tcgctatgca tcgatcattt gcaacgccac ggggccagca aaaaatccat
     7441 tacccgtgcg tttgatgacg atgttgagtt tcaggagcgc atggcagaac acatccggta
     7501 catggttgaa accattgctc accaccaggt tgatattgat tcagaggtat aaagaaggcc
     7561 atcctgacgg atggcctttt ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     7621 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattctca
     7681 tgtttgacag cttatcatcg ataagcttta atgcggtagt ttatcacagt taaattgcta
     7741 acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc tcggcaccgt
     7801 caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc tcttgcggga
     7861 tatcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc tatatgcgtt
     7921 gatgcaat
//
