LOCUS       sequence-255831-        4418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR template for genomic N-terminal Nui-HA tag, nat-CUP1p flanking 
            loxP sites allow expression by the native promoter after 
            CRE-recombinase expression, nat marker confers resistance to 
            Nourseothricin .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4418)
  AUTHORS   Dunkler A, Muller J, Johnsson N
  TITLE     Detecting protein-protein interactions with the Split-Ubiquitin 
            sensor.
  JOURNAL   Methods Mol Biol. 2012;786:115-30. doi: 10.1007/978-1-61779-292-2_7.
  PUBMED    21938623
REFERENCE   2  (bases 1 to 4418)
  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: "Methods Mol
            Biol."; date: "2012"; volume: "786:115-30. doi"; pages: " 
            10.1007/978-1-61779-292-2_7"
COMMENT     Sequence Label: pFA NuiHA-loxP-nat-CUP1p
FEATURES             Location/Qualifiers
     source          1..4418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        19..37
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     19..36
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             201..227
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    234..267
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        303..646
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             647..1219
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1227..1414
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1282..1306)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        1723..1957
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     protein_bind    1987..2020
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2090..2109)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2091..2109)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2196..2213)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2367..2955)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2447..2466)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3126..3917,3918..3986))
                     /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     3749..3768
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3987..4091)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4159..4177
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4215..4237)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4337..4356
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atgtatcata cacatacgat ttaggtgaca ctatagaacg cggccgccag ctgaagcttc
       61 gtacgctgca ggtcgacatg cagattttcg tcaagacttt gaccggtaaa accataacat
      121 tggaagttga atcttccgat accatcgaca acgttaagtc gaaaattcaa gacaaggaag
      181 ggatccctgg gtctggggct tacccatacg atgttcctga ctatgcgaga tccataactt
      241 cgtataatgt atgctatacg aagttatagc ttgcctcgtc cccgccgggt cacccggcca
      301 gcgacatgga ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat
      361 gatgtgactg tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca
      421 tacattttga tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cagacctgcg
      481 agcagggaaa cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga
      541 gaaatataaa aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa
      601 tcttgctagg atacagttct cacatcacat ccgaacataa acaaccatgg gtaccactct
      661 tgacgacacg gcttaccggt accgcaccag tgtcccgggg gacgccgagg ccatcgaggc
      721 actggatggg tccttcacca ccgacaccgt cttccgcgtc accgccaccg gggacggctt
      781 caccctgcgg gaggtgccgg tggacccgcc cctgaccaag gtgttccccg acgacgaatc
      841 ggacgacgaa tcggacgacg gggaggacgg cgacccggac tcccggacgt tcgtcgcgta
      901 cggggacgac ggcgacctgg cgggcttcgt ggtcgtctcg tactccggct ggaaccgccg
      961 gctgaccgtc gaggacatcg aggtcgcccc ggagcaccgg gggcacgggg tcgggcgcgc
     1021 gttgatgggg ctcgcgacgg agttcgcccg cgagcggggc gccgggcacc tctggctgga
     1081 ggtcaccaac gtcaacgcac cggcgatcca cgcgtaccgg cggatggggt tcaccctctg
     1141 cggcctggac accgccctgt acgacggcac cgcctcggac ggcgagcagg cgctctacat
     1201 gagcatgccc tgcccctaat ctcgaggcga atttcttatg atttatgatt tttattatta
     1261 aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac
     1321 gaaaattctt attcttgagt aaccctttcc tgtaggtcag gttgctttct caggtatagc
     1381 atgaggtcgc tcttattgac cacacctcta ccggcatgcc gagcaaatgc ctgcaaatcg
     1441 ctccccattt cacccaattg tagatatgct aactccagca atgagttgat gaatctcggt
     1501 gtgtatttta tgtcctcaga ggacaacacc tgttgtaatc gagctctagt aagccgatcc
     1561 cattaccgac atttgggcgc tatacgtgca tatgttcatg tatgtatctg tatttaaaac
     1621 acttttgtat tatttttcct catatatgtg tataggttta tacggatgat ttaattatta
     1681 cttcaccacc ctttatttcg ggctgatatc ttagccttgt tactagttag aaaaagacat
     1741 ttttgctgtc agtcactgtc aagagattct tttgctggca tttcttctag aagcaaaaag
     1801 agcgatgcgt cttttccgct gaaccgttcc agcaaaaaag actaccaacg caatatggat
     1861 tgtcagaatc atataaaaga gaagcaaata actccttgtc ttgtatcaat tgcattataa
     1921 tatcttcttg ttagtgcaat atcatataga agtcatcgaa atagatatta agaaaaacaa
     1981 actgtaataa cttcgtataa tgtatgctat acgaagttat tgacgacaga gaattcatcg
     2041 atgatatcag atccactagt ggcctatgcg gccgcggatc tgccggtctc cctatagtga
     2101 gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg
     2161 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2221 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2281 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2341 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2401 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2461 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2521 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2581 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2641 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2701 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2761 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     2821 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2881 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2941 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3001 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3061 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3121 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3181 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3241 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3301 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3361 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3421 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3481 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3541 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3601 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3661 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3721 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3781 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3841 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3901 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3961 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4021 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4081 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     4141 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     4201 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     4261 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     4321 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatggacata
     4381 ttgtcgttag aacgcggcta caattaatac ataacctt
//
