LOCUS       sequence-384242-        4210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pFA6a derived selection cassette flanked with transcription 
            terminators (tDEG1/tCYC1), allows genome modification without 
            disruption of insertion neighboring genes by transcription 
            interference.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4210)
  AUTHORS   Powers EN, Chan C, Doron-Mandel E, Llacsahuanga Allcca L, Kim Kim J,
            Jovanovic M, Brar GA
  TITLE     Bidirectional promoter activity from expression cassettes can drive 
            off-target repression of neighboring gene translation.
  JOURNAL   Elife. 2022 Dec 12;11:e81086. doi: 10.7554/eLife.81086.
  PUBMED    36503721
REFERENCE   2  (bases 1 to 4210)
  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: "Elife."; 
            date: "2022-12-12"; volume: "11:e81086. doi"; pages: " 
            10.7554/eLife.81086"
COMMENT     Sequence Label: kanMX6-ins2
FEATURES             Location/Qualifiers
     source          1..4210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..54
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     36..53
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     gene            217..1573
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        217..560
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             561..1370
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(628..647)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1376..1573
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(1645..1663)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1899..1918)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1900..1918)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2005..2022)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2176..2764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2256..2275)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2935..3726,3727..3795))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3558..3577
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3796..3900)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3968..3986
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4024..4046)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4146..4165
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aattaataca taaccttatg tatcatacac atacgattta ggtgacacta tagaacgcgg
       61 ccgccagctg aagcttcgta cgctgcaggt cgacggatcc ccgggttaat taagaaaaga
      121 aatatagtct tcaaggttat attatacagg tttatatatt attggcgcgc cagatctgtt
      181 tagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca gaataccctc
      241 cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag
      301 cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga
      361 agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc cctcacagac
      421 gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca
      481 ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag ttctcacatc
      541 acatccgaac ataaacaacc atgggtaagg aaaagactca cgtttcgagg ccgcgattaa
      601 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
      661 caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac
      721 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
      781 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
      841 tactcaccac tgcgatcccc ggcaaaacag cattccaggt attagaagaa tatcctgatt
      901 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
      961 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     1021 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     1081 aacaagtctg gaaagaaatg cataagcttt tgccattctc accggattca gtcgtcactc
     1141 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     1201 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     1261 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     1321 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagtactga
     1381 caataaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt
     1441 tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg
     1501 cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct
     1561 ggtcgctata ctgctgtcga ttcgatacta acgccgccat ccaacaggcc ccttttcctt
     1621 tgtcgatatc atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc
     1681 tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat
     1741 agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacaa
     1801 acgcgtgtac gcatgtaaca ttatagttta aacgagctcg aattcatcga tgatatcaga
     1861 tccactagtg gcctatgcgg ccgcggatct gccggtctcc ctatagtgag tcgtattaat
     1921 ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1981 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     2041 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     2101 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2161 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2221 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2281 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2341 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2401 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2461 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2521 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2581 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2641 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2701 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2761 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     2821 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     2881 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     2941 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3001 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3061 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3121 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3181 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3241 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3301 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3361 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3421 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3481 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     3541 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     3601 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     3661 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     3721 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     3781 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     3841 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     3901 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     3961 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     4021 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     4081 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     4141 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga
     4201 acgcggctac
//
