LOCUS       sequence-488087-        3108 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GoldenBraid2.0 Flp recombinase excisable antibiotic resistance 
            cassette in the grammar of an assembled transcription unit. Ready to
            be amplified to make gene knockouts..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3108)
  AUTHORS   Cooke MB, Welch KT, Deus Ramirez L, Wen AX, Marciano DC,, Herman C
  TITLE     GoldenBraid2.0 E. coli: A comprehensive and characterized toolkit 
            for enterics.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3108)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pR6Ko1_FRTkanFRT
FEATURES             Location/Qualifiers
     source          1..3108
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    40..73
                     /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)"
     CDS             441..1235
                     /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(495..514)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1105..1124
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1248..1295
                     /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)."
     promoter        complement(1386..1404)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1387..1404)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1422..1438)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1422..1438)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      1509..1581
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      1637..1993
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(2107..2766)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2681..2700
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2767..2869)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(2888..2915)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     3056..3073
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3057..3073
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3080..3099
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3080..3098
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggtctcagga gattccgggg atccgtcgac ctgcagttcg aagttcctat tctctagaaa
       61 gtataggaac ttcagagcgc ttttgaagct cacgctgccg caagcactca gggcgcaagg
      121 gctgctaaag gaagcggaac acgtagaaag ccagtccgca gaaacggtgc tgaccccgga
      181 tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg
      241 tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg
      301 aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact
      361 ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagatct gatcaagaga
      421 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
      481 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
      541 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
      601 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
      661 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
      721 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
      781 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
      841 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
      901 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
      961 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     1021 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     1081 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     1141 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     1201 tcgccttcta tcgccttctt gacgagttct tctaataagg ggatcttgaa gttcctattc
     1261 cgaagttcct attctctaga aagtatagga acttcgaagc agctccagcc tacacgctgt
     1321 caggaggatc gctctgccat ctagcacgct gtcatgagac cggatcctga cttgtattcc
     1381 gcggttctat agtgtcacct aaatagcttg gcgtaattta ggtcatagct gtttcctgtc
     1441 ggaccaaaac gaaaaaaggc ccccctttcg ggaggcctct tttctggaat ttggtaccga
     1501 gcaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     1561 gtttgtcggt tgaacgctct ctgacgtacc gcggccgcgt agaggatctg ttgatcagca
     1621 gttcaacctg ttgatagtac gtactaagct ctcatgtttc acgtactaag ctctcatgtt
     1681 taacgtacta agctctcatg tttaacgaac taaaccctca tggctaacgt actaagctct
     1741 catggctaac gtactaagct ctcatgtttc acgtactaag ctctcatgtt tgaacaataa
     1801 aattaatata aatcagcaac ttaaatagcc tctaaggttt taagttttat aagaaaaaaa
     1861 agaatatata aggcttttaa agcttttaag gtttaacggt tgtggacaac aagccaggga
     1921 tgtaacgcac tgagaagccc ttagagcctc tcaaagcaat tttgagtgac acaggaacac
     1981 ttaacggctg acatgtgatc atgtttgaca gcttatcatc gaatttctgc cattcatccg
     2041 cttattatca cttattcagg cgtagcaacc aggcgtttaa gggcaccaat aactgcctta
     2101 aaaaaattac gccccgccct gccactcgtc gcaatactgt tgtaattcat taagcattct
     2161 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
     2221 cttgtcgcct tgcgtatagt atttgcccat cgtgaaaacg ggggcgaaga agttgtccat
     2281 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg acacgaaaaa
     2341 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
     2401 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agaggcttga
     2461 aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
     2521 cagctcaccg tctttcattg ccatacgaaa ttccggatga gcattcatca ggcgggcaag
     2581 aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
     2641 cgtaatatcc agttgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
     2701 aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
     2761 ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag
     2821 tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcat aacattgtag
     2881 aaacgcaaaa aggccatccg tcaggatggc cttctgctta atttgatgcc tggcagttta
     2941 tggcgggcgt cctgcccgcc accctttcag ccaaaaaact taagaccgcc ggtcttgtcc
     3001 actaccttgc agtaatgcgg tggacaggat cggcggtttt cttttctctt ctcaatgtaa
     3061 aacgacggcc agtgaattgt aatacgactc actatagggt tcggatcc
//
