LOCUS       sequence-435435-        3725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR-associated transposases from V. cholerae and used for RNA 
            guided cargo insertion in Vibrio natriegens.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3725)
  AUTHORS   Xu J, Sun Y, Wu J, Yang S, Yang L
  TITLE     Chromosome recombination and modification by LoxP-mediated evolution
            in Vibrio natriegens using CRISPR-associated transposases.
  JOURNAL   Biotechnol Bioeng. 2024 Mar;121(3):1163-1172. doi: 
            10.1002/bit.28639. Epub 2023 Dec 22.
  PUBMED    38131162
REFERENCE   2  (bases 1 to 3725)
  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.28639";
            journalName: "Biotechnol Bioeng"; date: "2024-03"; volume: "121"; 
            issue: "3"; pages: "1163-1172"
COMMENT     Sequence Label: pDonor-LoxP
FEATURES             Location/Qualifiers
     source          1..3725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    211..244
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     383..405
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     terminator      complement(474..522)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     terminator      complement(584..632)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     terminator      complement(745..776)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     RBS             802..824
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    852..885
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(994..1809)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(994..1809)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1717..1736
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1810..1901)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2249..2471
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             2519..3469
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 gaagcggggt tttttgcgaa aaaaataggg ataacagggt aatgttaact gttgatgcaa
       61 ccataaagtg atatttaata attatttata atcagcaact taaccacaaa acaaccatat
      121 attgatatct cacaaaacaa ccataagttg atatttttgt gaatcgagta tttcagcaaa
      181 actactgcag taaggaccaa ttggatccga ataacttcgt ataatgtatg ctatacgaag
      241 ttatggggat cgagcccata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
      301 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
      361 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
      421 ccgaaacgcg cgaggcagag cttggaattg cgaataataa ttttttcacg ttgaaaatct
      481 ccaaaaaaaa aggctccaaa aggagccttt aattgtatcg gtttatcagc ttgctttcga
      541 ggtgaattag cttggaattg cgaataataa ttttttcacg ttgaaaatct ccaaaaaaaa
      601 aggctccaaa aggagccttt aattgtatcg gtttatcagc ttgctttcga ggtgaatttc
      661 gacctctaga ggatccccgg gtaccgagct cactagttta attaactcga gacgcgtaac
      721 ctcgcgcctt acctgttgag caatagtcaa aagcctccgg tcggaggctt ttgactttct
      781 gcttagctag ccatatgtat atctccttct taaagttaaa caaaattatt cctaggaagc
      841 ttatgcatgt cataacttcg tataatgtat gctatacgaa gttatgacca tggaattccc
      901 gtgggtgtga taattatcaa tttatgggtg taattatcat tttatggttg tatcaacagt
      961 ttaaacatta ccctgttatc cctgaattaa ttcttagaaa aactcatcga gcatcaaatg
     1021 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     1081 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     1141 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     1201 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     1261 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     1321 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcggtc
     1381 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     1441 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     1501 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     1561 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     1621 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     1681 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     1741 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     1801 atggctcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     1861 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggcatgc tagcgcacca
     1921 catagcagac ccgccataaa acgccctgag aagcccgtga cgggcttttc ttgtattatg
     1981 ggtagtttcc ttgcatgaat ccataaaagg cgcctgtagt gccatttacc cccattcact
     2041 gccagagccg tgagcgcagc gaactgaatg tcacgaaaaa gacagcgact caggtgcctg
     2101 atggtcggag acaaaaggaa tattcagcga tttgcccgag cttgcgaggg tgctacttaa
     2161 gcctttaggg ttttaaggtc tgttttgtag aggagcaaac agcgtttgcg acatcctttt
     2221 gtaatactgc ggaactgact aaagtagtga gttatacaca gggctgggat ctattctttt
     2281 tatctttttt tattctttct ttattctata aattataacc acttgaatat aaacaaaaaa
     2341 aacacacaaa ggtctagcgg aatttacaga gggtctagca gaatttacaa gttttccagc
     2401 aaaggtctag cagaatttac agatacccac aactcaaagg aaaaggacta gtaattatca
     2461 ttgactagcc catctcaatt ggtatagtga ttaaaatcac ctagaccaat tgagatgtat
     2521 gtctgaatta gttgttttca aagcaaatga actagcgatt agtcgctatg acttaacgga
     2581 gcatgaaacc aagctaattt tatgctgtgt ggcactactc aaccccacga ttgaaaaccc
     2641 tacaaggaaa gaacggacgg tatcgttcac ttataaccaa tacgctcaga tgatgaacat
     2701 cagtagggaa aatgcttatg gtgtattagc taaagcaacc agagagctga tgacgagaac
     2761 tgtggaaatc aggaatcctt tggttaaagg ctttgagatt ttccagtgga caaactatgc
     2821 caagttctca agcgaaaaat tagaattagt ttttagtgaa gagatattgc cttatctttt
     2881 ccagttaaaa aaattcataa aatataatct ggaacatgtt aagtcttttg aaaacaaata
     2941 ctctatgagg atttatgagt ggttattaaa agaactaaca caaaagaaaa ctcacaaggc
     3001 aaatatagag attagccttg atgaatttaa gttcatgtta atgcttgaaa ataactacca
     3061 tgagtttaaa aggcttaacc aatgggtttt gaaaccaata agtaaagatt taaacactta
     3121 cagcaatatg aaattggtgg ttgataagcg aggccgcccg actgatacgt tgattttcca
     3181 agttgaacta gatagacaaa tggatctcgt aaccgaactt gagaacaacc agataaaaat
     3241 gaatggtgac aaaataccaa caaccattac atcagattcc tacctacata acggactaag
     3301 aaaaacacta cacgatgctt taactgcaaa aattcagctc accagttttg aggcaaaatt
     3361 tttgagtgac atgcaaagta agtatgatct caatggttcg ttctcatggc tcacgcaaaa
     3421 acaacgaacc acactagaga acatactggc taaatacgga aggatctgag gttcttatgg
     3481 ctcttgtatc tatcagtgaa gcatcaagac taacaaacaa aagtagaaca actgttcacc
     3541 gttacatatc aaagggaaaa ctgtccatat gcacagatga aaacggtgta aaaaagatag
     3601 atacatcaga gcttttacga gtttttggtg cattcaaagc tgttcaccat gaacagatcg
     3661 acaatgtaac agatgaacag catgtaacac agatcccggt gcctaatgag cccagtcacg
     3721 acgtt
//
