LOCUS       sequence-437500-        7682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ORFtag viral retroviral vector containing a constitutively active 
            promoter and a TetR tag followed by a splice donor sequence in 
            Frame3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7682)
  TITLE     Proteome-scale tagging and functional screening in mammalian cells 
            by ORFtag
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7682)
  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            "Unpublished"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: ORFtag_TetR-SD_Frame3
FEATURES             Location/Qualifiers
     source          1..7682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     183..202
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     436..453
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(534..553)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(575..904)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      589..724
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     889..909
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     1045..1067
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        1521..2020
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1556..1579)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1953..1972
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2033..2827
                     /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(2087..2106)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2697..2716
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    join(2833..3406,3407..3409,3410..3418)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2999..3016)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3226..3245
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3434..3454
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4112..4147
                     /codon_start=1
                     /product="nanobody epitope tag corresponding to amino acids
                     16-27 of human beta-catenin (Traenkle et al., 2015)"
                     /label=BC2 tag
                     /translation="PDRKAAVSHWQQ"
     CDS             4151..4216
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(4417..4833)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(4433..4449)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(4465..4487)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    5034..5233
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             5296..5471
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     promoter        complement(5472..5674)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5501..5521)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5675..6054
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(6154..6172)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6240..6344
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6345..6413,6414..7205)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6563..6582)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7376..7682,1..282)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
       61 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      121 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      181 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      241 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      301 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      361 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      421 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
      481 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
      541 tgctctgatg ccgcatagtt aagccagtaa gctttttgca aaagcctagg cctccaaaaa
      601 agcctcctca ctacttctgg aatagctcag aggccgaggc ggcctcggcc tctgcataaa
      661 taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg
      721 ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca
      781 tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac taattgagat
      841 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac
      901 acacattcca cagccggatc tacactccgc tatcgctacg tgactgggtc atggctgcgc
      961 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     1021 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     1081 caccgaaacg cgcgaggcag ccagcttacc tcccggtggt gggtcggtgg tccctgggca
     1141 ggggtctccc gatcccggac gagcccccaa atgaaagacc cccgctgacg ggtagtcaat
     1201 cactcagagg agaccctccc aaggaacagc gagaccacaa gtcggatgca actgcaagag
     1261 ggtttattgg atacacgggt acccgggcga ctcagtcaat cggaggactg gcgccccgag
     1321 tgaggggttg tgctagcttg ccaaacctac aggtggggtc tttcattccc ccctttttct
     1381 ggagactaaa taaaatcttt tattttatcg atggtcaagt tccttctggt tctggctctg
     1441 ctcatcgtcc tgcaggaccg gacgtgactg gagttcagac gtgtgctctt ccgatctcat
     1501 gttaactcga gaattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg
     1561 cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca
     1621 ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc
     1681 ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac
     1741 gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca
     1801 atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg
     1861 ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg
     1921 ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc
     1981 tgccgcgctg ttctcctctt cctcatctcc gggcctttcg gaattcgcca ccatgattga
     2041 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     2101 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     2161 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     2221 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     2281 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     2341 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     2401 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     2461 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     2521 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     2581 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     2641 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     2701 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     2761 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     2821 cttctgacgc gtcccctctc cctccccccc cctaacgtta ctggccgaag ccgcttggaa
     2881 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat
     2941 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
     3001 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct
     3061 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc
     3121 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa
     3181 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc
     3241 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
     3301 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc
     3361 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatcg ccacaaccag
     3421 atctgccacc atgcccaaaa agaagaggaa agtgatgtct agactggaca agagcaaagt
     3481 cataaactct gctctggaat tactcaatga agtcggtatc gaaggcctga cgacaaggaa
     3541 actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga agaacaagcg
     3601 ggccctgctc gatgccctgg caatcgagat gctggacagg catcataccc acttctgccc
     3661 cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat tccgctgtgc
     3721 tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa cagagaaaca
     3781 gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct ccctggagaa
     3841 cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat tggaggatca
     3901 ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta tgcccccact
     3961 tctgagacaa gcaattgagc tgttcgacca tcagggagcc gaacctgcct tccttttcgg
     4021 cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg gcgggccgga
     4081 tatcggaggc ggatcaggtg gcggtagcgt ccccgatcgt aaggctgcgg tctctcattg
     4141 gcaacaggac gactataagg accacgacgg agactacaag gatcatgata ttgattacaa
     4201 agacgatgac gataaggcca ggtaagtaat gctttttttt ttctcgctct catttttcaa
     4261 aaacccacat aaaaattgag gaaagggaag aattgttttc tccttccagc acctcgtaat
     4321 ttgacccgac tgatggttcc cattagtcac ataaagctgt agtcaagtac agacgtcccg
     4381 cggcttcgat accgtcacga agcaacggat ccggccggcg cctagagaag gagtgagggc
     4441 tggataaagg gaggatcgag gcggggtcga acgaggaggt tcaaggggga gagacggggc
     4501 ggatggagga agaggaggcg gaggcttagg gtgtacaaag ggcttgaccc agggaggggg
     4561 gtcaaaagcc aaggcttccc aggtcacgat gtaggggacc tggtctgggt gtccatgcgg
     4621 gccaggtgaa aagaccttga tcttaacctg ggtgatgagg tctcggttaa aggtgccgtc
     4681 tcgcggccat ccgacgttaa aggttggcca ttctgcagag cagaaggtaa cccaacgtct
     4741 cttcttgaca tctaccgact ggttgtgagc gatccgctcg acatctttcc agtgacctaa
     4801 ggtcaaactt aagggagtgg taacagtctg gccctaattt tcagacaaat acagaaacac
     4861 agtcagacag agacaacaca gaacgatgct gcagcgctgc agcagacaag acgcgcggct
     4921 tcggttccaa accgaaagca aaaattcaga cggaggcggg aactgtttta ggttctcgtc
     4981 tcctaccaga accacatatc ctgacggggt cggattccac atcgactccc ttcctcaggt
     5041 cgggccacaa aaacggcccc caaagtccct gggacgtctc ccagggttgc ggccgggtgt
     5101 tcagaactcg tcagttccac cacgggtccg ccagatacag agctagttag ctaactagta
     5161 cagacgcagg cgcataacat caaacataga cactagacaa tcggacagac acagataagt
     5221 tgctggccag cttacctccc ggtggtgggt cggtggtccc tgggcagggg tctccaaatc
     5281 ccggacgagc ccccaaatga aagacccccg tcgtgggtag tcaatcactc agaggagacc
     5341 ctcccaagga acagcgagac cacgattcgg atgcaaacag caagaggctt tattgggaat
     5401 acgggtaccc gggcgacgca gtctatcgga agactggcgc gccgagtgag gggttgtggg
     5461 ctcttttatt gagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc
     5521 gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaca
     5581 aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc
     5641 cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt
     5701 agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg
     5761 gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta
     5821 ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta
     5881 ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg
     5941 gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat
     6001 gaactaatga ccccgtaatt gattactatt aataactagt caataatcaa tgtcaacatg
     6061 gcggtaatgt tggacatgag ccaatataaa tgtacatatt atgatatgga tacaacgtat
     6121 gcaatggcca agctaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     6181 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     6241 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6301 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6361 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6421 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6481 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6541 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     6601 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6661 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6721 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6781 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6841 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     6901 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6961 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     7021 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     7081 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     7141 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     7201 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     7261 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7321 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7381 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7441 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7501 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     7561 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7621 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7681 ag
//
