LOCUS       sequence-169653-        5263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal PCR tagging cassette for yeast codon optimized split 
            GFP1-10 with CDC42 promoter and KAR2 signal sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5263)
  AUTHORS   Smoyer CJ, Katta SS, Gardner JM, Stoltz L, McCroskey S, Bradford WD,
            McClain M, Smith SE, Slaughter BD, Unruh JR, Jaspersen SL
  TITLE     Analysis of membrane proteins localizing to the inner nuclear 
            envelope in living cells.
  JOURNAL   J Cell Biol. 2016 Nov 21;215(4):575-590. Epub 2016 Nov 9.
  PUBMED    27831485
REFERENCE   2  (bases 1 to 5263)
  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: "J Cell 
            Biol."; date: "2016-11-21"; volume: "215(4)"; pages: "575-590. Epub 
            2016 Nov 9"
COMMENT     Sequence Label: pSJ1726 (pFA6-NATMX-CDC42pr-KAR2SS-GFP1-10)
FEATURES             Location/Qualifiers
     source          1..5263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(64..168)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     236..254
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(292..314)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     414..433
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        514..532
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     514..531
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     gene            645..1764
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        645..988
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             989..1561
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1567..1764
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3430..3449)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3431..3449)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3536..3553)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3707..4295)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3787..3806)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4466..5257,5258..5263,1..63))
                     /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     5089..5108
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
       61 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
      121 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
      181 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
      241 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
      301 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
      361 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
      421 agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa
      481 ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaacgcggcc
      541 gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc gggttaatta aggcgcgcca
      601 gatctgttta gcttgccttg tccccgccgg gtcacccggc cagcgacatg gaggcccaga
      661 ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt
      721 acatttagcc catacatccc catgtataat catttgcatc catacatttt gatggccgca
      781 cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga aacgctcccc
      841 tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg
      901 atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt
      961 ctcacatcac atccgaacat aaacaaccat gggtaccact cttgacgaca cggcttaccg
     1021 gtaccgcacc agtgtcccgg gggacgccga ggccatcgag gcactggatg ggtccttcac
     1081 caccgacacc gtcttccgcg tcaccgccac cggggacggc ttcaccctgc gggaggtgcc
     1141 ggtggacccg cccctgacca aggtgttccc cgacgacgaa tcggacgacg aatcggacga
     1201 cggggaggac ggcgacccgg actcccggac gttcgtcgcg tacggggacg acggcgacct
     1261 ggcgggcttc gtggtcgtct cgtactccgg ctggaaccgc cggctgaccg tcgaggacat
     1321 cgaggtcgcc ccggagcacc gggggcacgg ggtcgggcgc gcgttgatgg ggctcgcgac
     1381 ggagttcgcc cgcgagcggg gcgccgggca cctctggctg gaggtcacca acgtcaacgc
     1441 accggcgatc cacgcgtacc ggcggatggg gttcaccctc tgcggcctgg acaccgccct
     1501 gtacgacggc accgcctcgg acggcgagca ggcgctctac atgagcatgc cctgccccta
     1561 atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt atagtttttt
     1621 tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt ttttcgcctc
     1681 gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt
     1741 atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca tccagtgtcg
     1801 aaaacgagct cgccgctcta gacaggccgg aactcaaaag ggtaatttcg tgaaaaacaa
     1861 tcatctacgg tataaataac aatttaattt acgtctcttt cgaaaatgct cgtgtagctc
     1921 agtggttaga gcttcgtgct tatagcaaca ttcggtttcc gaagtttctg tgccaaagac
     1981 ctttcaaaca ggcctttaaa agcaacgcga cccgtcgtgg gttcaatccc cacctcgagc
     2041 actttctctt ttttttttaa aaaaagttgc attatttcta tcaactgttg tagctacgtt
     2101 acacctaatt ttgtattgta gactgttaga taatccacat atacaggtaa atacggcaca
     2161 ttgtcttcta aacatcgcat taaagatgtc ttccaccgtc gattcaaggg tcattcaata
     2221 cgggtaatac tgcttccatt gggcccttcc aaacgaaaca agatacggga aaaagtgata
     2281 aaaaaaagca aacaaggttg attgatcgat gatagacgcg attcttggaa aaggcaagag
     2341 cagcgttttg ataaataggt tccaggtgta ccgagatata ctgattatcc ttctctgtca
     2401 ttcttcactt tttgcaatag gtttcctttg tcgcaaaaaa ttatattcgt tatttattat
     2461 actattctat tttcctgagg agataggtta acaaacgaat tagagaagca aaacttataa
     2521 aacaagaaat aaacgtatta gctcttccac aaaggaactg ttttactttt ttaaagtccc
     2581 caagagtagt ctcaagggaa aaagcgtatc aaacatacca tgtttttcaa cagactaagc
     2641 gctggcaagc tgctggtacc actctccgtg gtcctgtacg cccttttcgt ggtaatatta
     2701 cctttatcca tgtctaaagg tgaagaattg tttactggtg ttgttccaat tttggttgaa
     2761 ttggatggtg atgttaatgg tcataaattt tctgttagag gtgaaggtga aggtgatgct
     2821 actattggta aattgacttt gaaatttatt tgtactactg gtaaattgcc agttccatgg
     2881 ccaactttgg ttactacttt gacttatggt gttcaatgtt tttctagata tccagatcat
     2941 atgaaacaac atgatttttt taaatctgct atgccagaag gttatgttca agaaagaact
     3001 atttctttta aagatgatgg taaatataaa actagagctg ttgttaaatt tgaaggtgat
     3061 actttggtta atagaattga attgaaaggt actgatttta aagaagatgg taatattttg
     3121 ggtcataaat tggaatataa ttttaattct cataatgttt atattactgc tgataaacaa
     3181 aaaaatggta ttaaagctaa ttttactgtt agacataatg ttgaagatgg ttctgttcaa
     3241 ttggctgatc attatcaaca aaatactcca attggtgatg gtccagtttt gttgccagat
     3301 aatcattatt tgtctactca aactgttttg tctaaagatc caaatgaaaa aggtggtggt
     3361 tctggtggtg gttctcatcg atgatatcag atccactagt ggcctatgcg gccgcggatc
     3421 tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat
     3481 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3541 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3601 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3661 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3721 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3781 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3841 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3901 aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3961 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4021 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4081 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4141 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4201 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4261 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4321 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4381 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4441 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4501 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4561 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4621 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4681 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4741 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4801 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4861 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4921 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4981 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5041 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5101 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5161 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5221 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaa
//
