LOCUS       sequence-404791-        3777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrating plasmid for ORBIT in E. coli with sacB for counter 
            selection. Used for markerless or scarless modifications..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3777)
  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 3777)
  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_attP1_sacB_kanR
FEATURES             Location/Qualifiers
     source          1..3777
                     /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             join(610..696,697..2031)
                     /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"
     CDS             2259..3053
                     /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(2313..2332)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2923..2942
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      3280..3668
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(3712..3745)
                     /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 gcggtctcag tggtgtacgg tacaaaccgt gtaggctgga
       61 gctgcttcga agttcctata ctttctagag aataggaact tcggaatagg aacttcaaga
      121 tcccccacgc tgccgcaagc actcagggcg caagggctgc taaaggaagc ggaacacgta
      181 gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact ggatgttaca
      241 ttgcacaaga tatgcaacag aaactataaa aatacagaga atgaaaagaa acagatagat
      301 tttttagttc tttaggcccg tagtctgcaa atccttttat gattttctat caaacaaaag
      361 aggaaaatag accagttgca atccaaacga gagtctaata gaatgaggtc gaaaagtaaa
      421 tcgcgcgggt ttgttactga taaagcaggc aagacctaaa atgtgtaaag ggcaaagtgt
      481 atactttggc gtcacccctt acatatttta ggtctttttt tattgtgcgt aactaacttg
      541 ccatcttcaa acaggagggc tggaagaagc agaccgctaa cacagtacat aaaaaaggag
      601 acatgaacga tgaacatcaa aaagtttgca aaacaagcaa cagtattaac ctttactacc
      661 gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca aaagccatat
      721 aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat ccctgaacag
      781 caaaaaaatg aaaaatatca agttcctgaa ttcgattcgt ccacaattaa aaatatctct
      841 tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacgctga cggcactgtc
      901 gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa aaatgcggat
      961 gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa
     1021 aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc tatcctaaaa
     1081 gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa aatccgttta
     1141 ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac tgcacaagtt
     1201 aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta taaatcaatc
     1261 tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga aggcaactac
     1321 agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa aggccacaaa
     1381 tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga agaatcttta
     1441 tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt
     1501 ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg tatgattgag
     1561 ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc taacacagta
     1621 acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta cctgttcact
     1681 gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat ttacatgctt
     1741 ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac tggccttgtg
     1801 ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt cgctgtacct
     1861 caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg attctacgca
     1921 gacaaacaat caacgtttgc gccaagcttc ctgctgaaca tcaaaggcaa gaaaacatct
     1981 gttgtcaaag acagcatcct tgaacaagga caattaacag ttaacaaata agcgtcagac
     2041 gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta gactgggcgg
     2101 ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt aaggttggga
     2161 agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat
     2221 caagatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     2281 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2341 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2401 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     2461 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     2521 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     2581 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     2641 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     2701 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     2761 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     2821 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     2881 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     2941 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3001 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     3061 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     3121 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     3181 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccagct tcaaaagcgc
     3241 tctaaggagg atattcatat ggaccatggc taattcccat gtcagccgtt aagtgttcct
     3301 gtgtcactca aaattgcttt gagaggctct aagggcttct cagtgcgtta catccctggc
     3361 ttgttgtcca caaccgttaa accttaaaag ctttaaaagc cttatatatt cttttttttc
     3421 ttataaaact taaaacctta gaggctattt aagttgctga tttatattaa ttttattgtt
     3481 caaacatgag agcttagtac gtgaaacatg agagcttagt acgttagcca tgagagctta
     3541 gtacgttagc catgagggtt tagttcgtta aacatgagag cttagtacgt taaacatgag
     3601 agcttagtac gtgaaacatg agagcttagt acgtactatc aacaggttga actgctgatc
     3661 ttcagatcct ctacgccgga cgcatcgtgg ccggatcttg cggccgcaaa agaagttcct
     3721 atactttcta gagaatagga acttcggaat aggaactatt acacgtcttg agcgatt
//
