LOCUS       sequence-476310-        4237 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Electroporation vector for expression of Cre recombinase in 
            Cuprividus necator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4237)
  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 4237)
  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: pCnCre
FEATURES             Location/Qualifiers
     source          1..4237
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(60..648)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(140..159)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             803..1414
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     terminator      complement(1415..1462)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        join(1480..1485,1486..1501,1502..1508)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1514..1541
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     RBS             1586..1608
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1616..2647
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     primer_bind     complement(2694..2711)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2864..2950
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3042..3069
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3253..3355
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3356..4015
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3422..3441)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
       61 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      121 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      181 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
      241 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
      301 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
      361 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
      421 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
      481 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
      541 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
      601 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
      661 atcttttcta cggggtcgac acggatggcc tttttgcgtt tctacaaact cttttgttta
      721 tttttctaaa tacattcaaa tatgtatccg ctcatgaaca ataaaactgt ctgcttacat
      781 aaaacattga aaaaggaaga gtatgagtcc aaagagaaga acacaggcag agcgcgcaat
      841 ggagacccag ggcaagttga ttgcagcggc cctgggggtt ttacgggaaa aaggttacgc
      901 gggattccgg atcgcagatg tgcccggtgc tgcaggtgtc tcgagaggag cgcagagcca
      961 tcatttcccg acaaagcttg agcttctgct tgccactttt gaatggcttt acgaacagat
     1021 caccgaacgc agtcgggctc gattagcgaa attgaagcca gaggatgacg tcatccagca
     1081 aatgctggac gacgccgccg aatttttcct cgacgatgac ttctctatca gccttgattt
     1141 gattgtggct gccgaccggg atccagcgtt acgcgagggt attcagcgca cggtagagag
     1201 gaatcggttt gtcgtcgagg atatgtggct tggtgttctg gtgagccgtg gtctttcgcg
     1261 tgatgatgca gaagatatcc tttggttgat attcaattcg gtgcgtgggc ttgctgttcg
     1321 tagcctatgg cagaaggaca aagaacgctt tgagcgtgtc aggaactcga cactcgaaat
     1381 tgcgcgagag cggtacgcga aattcaagcg ctagcaaaaa acccctcaag acccgtttag
     1441 aggccccaag gggttatgct aggcggccgc aatgagctgt tgacaattaa tcatcggctc
     1501 gtataatgtg tggaacaaac agacaatctg gtctgtttgt attatagaat tcgagctcgg
     1561 tacccgggga tcctctagaa ataattttgt ttaactttaa gaaggagata tacatatgtc
     1621 caatttactg accgtacacc aaaatttgcc tgcattaccg gtcgatgcaa cgagtgatga
     1681 ggttcgcaag aacctgatgg acatgttcag ggatcgccag gcgttttctg agcatacctg
     1741 gaaaatgctt ctgtccgttt gccggtcgtg ggcggcatgg tgcaagttga ataaccggaa
     1801 atggtttccc gcagaacctg aagatgttcg cgattatctt ctatatcttc aggcgcgcgg
     1861 tctggcagta aaaactatcc agcaacattt gggccagcta aacatgcttc atcgtcggtc
     1921 cgggctgcca cgaccaagtg acagcaatgc tgtttcactg gttatgcggc ggatccgaaa
     1981 agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta gcgttcgaac gcactgattt
     2041 cgaccaggtt cgttcactca tggaaaatag cgatcgctgc caggatatac gtaatctggc
     2101 atttctgggg attgcttata acaccctgtt acgtatagcc gaaattgcca ggatcagggt
     2161 taaagatatc tcacgtactg acggtgggag aatgttaatc catattggca gaacgaaaac
     2221 gctggttagc accgcaggtg tagagaaggc acttagcctg ggggtaacta aactggtcga
     2281 gcgatggatt tccgtctctg gtgtagctga tgatccgaat aactacctgt tttgccgggt
     2341 cagaaaaaat ggtgttgccg cgccatctgc caccagccag ctatcaactc gcgccctgga
     2401 agggattttt gaagcaactc atcgattgat ttacggcgct aaggatgact ctggtcagag
     2461 atacctggcc tggtctggac acagtgcccg tgtcggagcc gcgcgagata tggcccgcgc
     2521 tggagtttca ataccggaga tcatgcaagc tggtggctgg accaatgtaa atattgtcat
     2581 gaactatatc cgtaacctgg atagtgaaac aggggcaatg gtgcgcctgc tggaagatgg
     2641 cgattagaag cttatttaaa tggctgtttt ggcggatgag agaagatttt cagcctgata
     2701 cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc
     2761 gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt
     2821 agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc
     2881 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     2941 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
     3001 ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga
     3061 tggccttttt gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt
     3121 atccgctcat gaagcggccg ctcatgacga ataaatacct gtgacggaag atcacttcgc
     3181 agaataaata aatcctggtg tccctgttga taccgggaag ccctgggcca acttttggcg
     3241 aaaatgagac gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc
     3301 actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag ctaaaatgga
     3361 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     3421 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     3481 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
     3541 tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct
     3601 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt
     3661 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca
     3721 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat
     3781 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa
     3841 tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa
     3901 ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg
     3961 cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaattttt
     4021 ttaaggcagt tattggtgcc cttaaacgcc tggttgctac gcctgaataa gtgataataa
     4081 gcggatgaat ggcagaaatt cgaaagcaaa ttcgacccgg tcgtcggttc agggcagggt
     4141 cgttaaatag ccgcttatgt ctattgctgg tttaccggtt tattgactac cggaagcagt
     4201 gtgaccgtgt gcttctcaaa tgcctgaggc cagttaa
//
