LOCUS       sequence-404790-        5386 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrating plasmid for ORBIT in E. coli with self excising factor 
            (Xis) and sacB for counter selection. Used for markerless 
            modifications..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5386)
  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 5386)
  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_tsXis_sacB_kanR
FEATURES             Location/Qualifiers
     source          1..5386
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1255..1341,1342..2676)
                     /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             2904..3698
                     /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(2958..2977)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3568..3587
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      3925..4313
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(4357..4390)
                     /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)"
     protein_bind    4423..4470
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    complement(4491..4538)
                     /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             complement(4654..5367)
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
ORIGIN
        1 aagggataaa tatctaacac cgtgcgtgtt gactatttta cctctggcgg tgataatggt
       61 tgcatgtact aaccttaaca aactacggag gtcaacatgg ccggcactca gcgtatcgtc
      121 tttctacccg atactcagtt gcctttcgag gcgcgcaaag agatgcaagc ggtcatccgc
      181 ttcatcgggg atgtccagcc gtacggcgtg gtacatatcg gtgacgtcct agacctgccg
      241 cagccctcgc gctggaaccg gggaaccaag ggcgagttcg agggctcggt gtaccgcgac
      301 gcggactacg ccaagaagaa cctgatggag ccgctgcgca aggtctacga cggctggatc
      361 gggatgcacg agggcaacca cgatctgcga gcccgcgagt acctggccaa gaacgcaccg
      421 gccctggagg gtacgcacgc tttcgacatc gacgtgctgc tcgacttcga cgggttcggt
      481 gtggagctgc tgcctgactt ctacgacatc gctccgggct ggatctccac tcacgggcac
      541 atgggcaaga tgacgctatc ccagatcgcc ggatcgacag cgctcaacgg tgccaagaag
      601 ttcggcaagt ccgtggtctg cggccacacg caccggcagg ctgtcgtctc gcactcgttc
      661 gggtacggcg gctcggtgcg caagaccgtc accggcatgg aagtcgggca cctgatggac
      721 atgaagaagg ccaactatct aaagggcgga gccgggaact ggcagatggg cttcgggatg
      781 ctcacggtcg acggcaagca tgtcaaggct gagatcgtcc cgatcctggg aggcaagttc
      841 accgttgacg gccaggtctg ggaagtcacc ggttaagatg ttacattgca caagatatgc
      901 aacagaaact ataaaaatac agagaatgaa aagaaacaga tagatttttt agttctttag
      961 gcccgtagtc tgcaaatcct tttatgattt tctatcaaac aaaagaggaa aatagaccag
     1021 ttgcaatcca aacgagagtc taatagaatg aggtcgaaaa gtaaatcgcg cgggtttgtt
     1081 actgataaag caggcaagac ctaaaatgtg taaagggcaa agtgtatact ttggcgtcac
     1141 cccttacata ttttaggtct ttttttattg tgcgtaacta acttgccatc ttcaaacagg
     1201 agggctggaa gaagcagacc gctaacacag tacataaaaa aggagacatg aacgatgaac
     1261 atcaaaaagt ttgcaaaaca agcaacagta ttaaccttta ctaccgcact gctggcagga
     1321 ggcgcaactc aagcgtttgc gaaagaaacg aaccaaaagc catataagga aacatacggc
     1381 atttcccata ttacacgcca tgatatgctg caaatccctg aacagcaaaa aaatgaaaaa
     1441 tatcaagttc ctgaattcga ttcgtccaca attaaaaata tctcttctgc aaaaggcctg
     1501 gacgtttggg acagctggcc attacaaaac gctgacggca ctgtcgcaaa ctatcacggc
     1561 taccacatcg tctttgcatt agccggagat cctaaaaatg cggatgacac atcgatttac
     1621 atgttctatc aaaaagtcgg cgaaacttct attgacagct ggaaaaacgc tggccgcgtc
     1681 tttaaagaca gcgacaaatt cgatgcaaat gattctatcc taaaagacca aacacaagaa
     1741 tggtcaggtt cagccacatt tacatctgac ggaaaaatcc gtttattcta cactgatttc
     1801 tccggtaaac attacggcaa acaaacactg acaactgcac aagttaacgt atcagcatca
     1861 gacagctctt tgaacatcaa cggtgtagag gattataaat caatctttga cggtgacgga
     1921 aaaacgtatc aaaatgtaca gcagttcatc gatgaaggca actacagctc aggcgacaac
     1981 catacgctga gagatcctca ctacgtagaa gataaaggcc acaaatactt agtatttgaa
     2041 gcaaacactg gaactgaaga tggctaccaa ggcgaagaat ctttatttaa caaagcatac
     2101 tatggcaaaa gcacatcatt cttccgtcaa gaaagtcaaa aacttctgca aagcgataaa
     2161 aaacgcacgg ctgagttagc aaacggcgct ctcggtatga ttgagctaaa cgatgattac
     2221 acactgaaaa aagtgatgaa accgctgatt gcatctaaca cagtaacaga tgaaattgaa
     2281 cgcgcgaacg tctttaaaat gaacggcaaa tggtacctgt tcactgactc ccgcggatca
     2341 aaaatgacga ttgacggcat tacgtctaac gatatttaca tgcttggtta tgtttctaat
     2401 tctttaactg gcccatacaa gccgctgaac aaaactggcc ttgtgttaaa aatggatctt
     2461 gatcctaacg atgtaacctt tacttactca cacttcgctg tacctcaagc gaaaggaaac
     2521 aatgtcgtga ttacaagcta tatgacaaac agaggattct acgcagacaa acaatcaacg
     2581 tttgcgccaa gcttcctgct gaacatcaaa ggcaagaaaa catctgttgt caaagacagc
     2641 atccttgaac aaggacaatt aacagttaac aaataagcgt cagacgcgca aagagaaagc
     2701 aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa
     2761 gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa
     2821 actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag
     2881 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     2941 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3001 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3061 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3121 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3181 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3241 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3301 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3361 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3421 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3481 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3541 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3601 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3661 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     3721 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     3781 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3841 ggatctcatg ctggagttct tcgcccaccc cagcttcaaa agcgctctaa ggaggatatt
     3901 catatggacc atggctaatt cccatgtcag ccgttaagtg ttcctgtgtc actcaaaatt
     3961 gctttgagag gctctaaggg cttctcagtg cgttacatcc ctggcttgtt gtccacaacc
     4021 gttaaacctt aaaagcttta aaagccttat atattctttt ttttcttata aaacttaaaa
     4081 ccttagaggc tatttaagtt gctgatttat attaatttta ttgttcaaac atgagagctt
     4141 agtacgtgaa acatgagagc ttagtacgtt agccatgaga gcttagtacg ttagccatga
     4201 gggtttagtt cgttaaacat gagagcttag tacgttaaac atgagagctt agtacgtgaa
     4261 acatgagagc ttagtacgta ctatcaacag gttgaactgc tgatcttcag atcctctacg
     4321 ccggacgcat cgtggccgga tcttgcggcc gcaaaagaag ttcctatact ttctagagaa
     4381 taggaacttc ggaataggaa ctattacacg tcttgagcga ttggtttgtc tggtcaacca
     4441 ccgcggtctc agtggtgtac ggtacaaacc gtgtaggctg gagctgcttc gaagttccta
     4501 tactttctag agaataggaa cttcggaata ggaacttcaa gatcccccac gctgccgcaa
     4561 gcactcaggg cgcaagggct gctaaaggaa gcggaacacg tagaaagcca gtccgcagaa
     4621 acggtgctga ccccggatga atgtcagcta ctgtcagcca aacgtctctt caggccactg
     4681 actagcgata actttcccca caacggaaca actctcattg catgggatca ttgggtactg
     4741 tgggtttagt ggttgtaaaa acacctgacc gctatccctg atcagtttct tgaaggtaaa
     4801 ctcatcaccc ccaagtctgg ctatgcagaa atcacctggc tcaacagcct gctcagggtc
     4861 aacgagaatt aacattccgt caggaaagct tggcttggag cctgttggtg cggtcatgga
     4921 attaccttca acctcaagcc agaatgcaga atcactggct tttttggttg tgcttaccca
     4981 tctctccgca tcacctttgg taaaggttct aagcttaggt gagaacatcc ctgcctgaac
     5041 atgagaaaaa acagggtact catactcact tctaagtgac ggctgcatac taaccgcttc
     5101 atacatctcg tagatttctc tggcgattga agggctaaat tcttcaacgc taactttgag
     5161 aatttttgta agcaatgcgg cgttataagc atttaatgca ttgatgccat taaataaagc
     5221 accaacgcct gactgcccca tccccatctt gtctgcgaca gattcctggg ataagccaag
     5281 ttcatttttc tttttttcat aaattgcttt aaggcgacgt gcgtcctcaa gctgctcttg
     5341 tgttaatggt ttcttttttg tgctcatacg ttaaatctat caccgc
//
