LOCUS       sequence-169656-        6428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ONM/ER split GFP1-10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6428)
  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 6428)
  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: pSJ2039 (pRS316-NOP1pr-GFP1-10-SCS2TM)
FEATURES             Location/Qualifiers
     source          1..6428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        196..416
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             417..1220
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1351..1806)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1488..1509)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1700..1719
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1936..1958
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1950..1967
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1951..1967
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1974..1993
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1974..1992
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3370..3535
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3425..3449)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     3558..3574
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3558..3574
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3608..3624)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3609..3625)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3654..3672)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3693..3709)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3693..3709)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3706..3728)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3717..3733
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3741..3747,3748..3765,3766..3771))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3924..3941)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4095..4683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4175..4194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4854..5645,5646..5714))
                     /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     5477..5496
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5715..5819)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5856..6359
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6401..6419
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
      241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
      961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
     1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
     1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg
     1981 actcactata gggcgaattg gagctcaacc ggaacttgtt tagattcctt tttgtaggtt
     2041 aatcttatcc tgtacttgct gatattgtgg ttattagcag agattttttt caaaacatca
     2101 tctaaagtag gctttttgga taagtcaatt tcagtgtccc ttaacccttt agagcggctt
     2161 tttatggtga taggcatttt caaattaaat tcaaaatatg tatctctctc aaatagctca
     2221 attggtttct agatagctaa atagaatata atcttactgt cctccgttct gtaaaattca
     2281 cgctcttagt cccttttcat aattccttaa ctttttgcgt acaaaatgat atgtttatta
     2341 tatttttctt tttttttttt caaatttttt ctttttcttg aaaaattttt caaattggaa
     2401 agctcatctc tcttgaatgt ataatacttt cttcctctaa ctttcaaaaa gttttacata
     2461 gccaagaagt tttccttaca tcggtatact actgttatat aagttattct tcgagaaaca
     2521 attagatatc attcatcgga taaatctaag ttgcccattg ctttcaataa ctccgatcaa
     2581 attaactcaa atcaactaaa acagtagatc catgtctaaa ggtgaagaat tgtttactgg
     2641 tgttgttcca attttggttg aattggatgg tgatgttaat ggtcataaat tttctgttag
     2701 aggtgaaggt gaaggtgatg ctactattgg taaattgact ttgaaattta tttgtactac
     2761 tggtaaattg ccagttccat ggccaacttt ggttactact ttgacttatg gtgttcaatg
     2821 tttttctaga tatccagatc atatgaaaca acatgatttt tttaaatctg ctatgccaga
     2881 aggttatgtt caagaaagaa ctatttcttt taaagatgat ggtaaatata aaactagagc
     2941 tgttgttaaa tttgaaggtg atactttggt taatagaatt gaattgaaag gtactgattt
     3001 taaagaagat ggtaatattt tgggtcataa attggaatat aattttaatt ctcataatgt
     3061 ttatattact gctgataaac aaaaaaatgg tattaaagct aattttactg ttagacataa
     3121 tgttgaagat ggttctgttc aattggctga tcattatcaa caaaatactc caattggtga
     3181 tggtccagtt ttgttgccag ataatcatta tttgtctact caaactgttt tgtctaaaga
     3241 tccaaatgaa aaaggtggtg gttctggtgg tggttctggg gcccaaaatg aatcatccag
     3301 catgggtata ttcatattgg ttgcactcct tatcttggtt ttaggatggt tctacagata
     3361 attaattaag cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa
     3421 ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg
     3481 agtaactctt tcctgtaggt caggttgctt tctcaggtat agtatgaggt cgctcttatt
     3541 gaccacaccg cggccgctct agaactagtg gatcccccgg gctgcaggaa ttcgatatca
     3601 agcttatcga taccgtcgac ctcgaggggg ggcccggtac ccagcttttg ttccctttag
     3661 tgagggttaa ttccgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3721 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3781 gcctaatgag tgaggtaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3841 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3901 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3961 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4021 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4081 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4141 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4201 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4261 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4321 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4381 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4441 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4501 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     4561 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4621 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4681 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4741 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4801 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4861 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4921 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4981 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5041 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5101 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5161 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5221 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5281 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5341 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5401 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5461 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5521 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5581 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5641 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5701 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5761 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5821 acatttcccc gaaaagtgcc acctgggtcc ttttcatcac gtgctataaa aataattata
     5881 atttaaattt tttaatataa atatataaat taaaaataga aagtaaaaaa agaaattaaa
     5941 gaaaaaatag tttttgtttt ccgaagatgt aaaagactct agggggatcg ccaacaaata
     6001 ctacctttta tcttgctctt cctgctctca ggtattaatg ccgaattgtt tcatcttgtc
     6061 tgtgtagaag accacacacg aaaatcctgt gattttacat tttacttatc gttaatcgaa
     6121 tgtatatcta tttaatctgc ttttcttgtc taataaatat atatgtaaag tacgcttttt
     6181 gttgaaattt tttaaacctt tgtttatttt tttttcttca ttccgtaact cttctacctt
     6241 ctttatttac tttctaaaat ccaaatacaa aacataaaaa taaataaaca cagagtaaat
     6301 tcccaaatta ttccatcatt aaaagatacg aggcgcgtgt aagttacagg caagcgatcc
     6361 gtcctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     6421 ctttcgtc
//
