LOCUS       sequence-415623-        1994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrating plasmid for ORBIT in E. coli. Orthogonal attP site 
            (#2)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 1994)
  AUTHORS   Saunders SH, Ahmed AM
  TITLE     ORBIT for E. coli: Kilobase-scale oligonucleotide recombineering at 
            high throughput and high efficiency
  JOURNAL   bioRxiv 2023
REFERENCE   2  (bases 1 to 1994)
  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: "bioRxiv 
            2023"
COMMENT     Sequence Label: pInt_attP2_kanR
FEATURES             Location/Qualifiers
     source          1..1994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..48
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    complement(69..116)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             476..1270
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(530..549)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1140..1159
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      1497..1885
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(1929..1962)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
ORIGIN
        1 ggtttgtctg gtcaaccacc gcgttctcag tggtgtacgg tacaaaccgt gtaggctgga
       61 gctgcttcga agttcctata ctttctagag aataggaact tcggaatagg aacttcaaga
      121 tcccccacgc tgccgcaagc actcagggcg caagggctgc taaaggaagc ggaacacgta
      181 gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg
      241 gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg
      301 atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc
      361 ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat
      421 ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat
      481 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
      541 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
      601 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
      661 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
      721 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
      781 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
      841 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
      901 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
      961 tcaggggctc gcgccagccg aactgttcgc taggctcaag gcgcgcatgc ccgacggcga
     1021 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     1081 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     1141 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     1201 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     1261 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     1321 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     1381 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     1441 cccagcttca aaagcgctct aaggaggata ttcatatgga ccatggctaa ttcccatgtc
     1501 agccgttaag tgttcctgtg tcactcaaaa ttgctttgag aggctctaag ggcttctcag
     1561 tgcgttacat ccctggcttg ttgtccacaa ccgttaaacc ttaaaagctt taaaagcctt
     1621 atatattctt ttttttctta taaaacttaa aaccttagag gctatttaag ttgctgattt
     1681 atattaattt tattgttcaa acatgagagc ttagtacgtg aaacatgaga gcttagtacg
     1741 ttagccatga gagcttagta cgttagccat gagggtttag ttcgttaaac atgagagctt
     1801 agtacgttaa acatgagagc ttagtacgtg aaacatgaga gcttagtacg tactatcaac
     1861 aggttgaact gctgatcttc agatcctcta cgccggacgc atcgtggccg gatcttgcgg
     1921 ccgcaaaaga agttcctata ctttctagag aataggaact tcggaatagg aactattaca
     1981 cgtcttgagc gatt
//
