LOCUS       sequence-437118-        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 
            Frame2.
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  JOURNAL   Unpublished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            "Unpublished"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_Frame2
FEATURES             Location/Qualifiers
     source          1..7682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     143..162
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     396..413
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(494..513)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(535..864)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      549..684
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     849..869
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     1005..1027
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        1481..1980
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1516..1539)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1913..1932
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1993..2787
                     /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(2047..2066)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2657..2676
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    join(2793..3366,3367..3369,3370..3378)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2959..2976)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3186..3205
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3394..3414
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4072..4107
                     /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             4111..4176
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(4376..4792)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(4392..4408)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(4424..4446)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    4993..5192
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             5255..5430
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     promoter        complement(5431..5634)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5460..5480)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5635..6014
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(6114..6132)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6200..6304
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6305..6373,6374..7165)
                     /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(6523..6542)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7336..7682,1..242)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
       61 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      121 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      181 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      241 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      301 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
      361 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
      421 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
      481 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtaa gctttttgca
      541 aaagcctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc
      601 ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga
      661 actgggcgga gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta
      721 attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct
      781 ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga
      841 ctttccacac cctaactgac acacattcca cagccggatc tacactccgc tatcgctacg
      901 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
      961 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     1021 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ccagcttacc tcccggtggt
     1081 gggtcggtgg tccctgggca ggggtctccc gatcccggac gagcccccaa atgaaagacc
     1141 cccgctgacg ggtagtcaat cactcagagg agaccctccc aaggaacagc gagaccacaa
     1201 gtcggatgca actgcaagag ggtttattgg atacacgggt acccgggcga ctcagtcaat
     1261 cggaggactg gcgccccgag tgaggggttg tgctagcttg ccaaacctac aggtggggtc
     1321 tttcattccc ccctttttct ggagactaaa taaaatcttt tattttatcg atggtcaagt
     1381 tccttctggt tctggctctg ctcatcgtcc tgcaggaccg gacgtgactg gagttcagac
     1441 gtgtgctctt ccgatctcat gttaactcga gaattctacc gggtagggga ggcgcttttc
     1501 ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
     1561 ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt
     1621 ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg
     1681 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
     1741 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
     1801 ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
     1861 aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
     1921 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg
     1981 gaattcgcca ccatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     2041 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     2101 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     2161 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     2221 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     2281 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     2341 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     2401 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     2461 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     2521 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     2581 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     2641 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     2701 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     2761 tatcgccttc ttgacgagtt cttctgacgc gtcccctctc cctccccccc cctaacgtta
     2821 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca
     2881 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca
     2941 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg
     3001 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc
     3061 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
     3121 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag
     3181 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
     3241 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt
     3301 taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat
     3361 gataatatcg ccacaaccag atctgccacc atgcccaaaa agaagaggaa agtgatgtct
     3421 agactggaca agagcaaagt cataaactct gctctggaat tactcaatga agtcggtatc
     3481 gaaggcctga cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac
     3541 tggcacgtga agaacaagcg ggccctgctc gatgccctgg caatcgagat gctggacagg
     3601 catcataccc acttctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac
     3661 gccaagtcat tccgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc
     3721 acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag
     3781 caaggcttct ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg
     3841 ggctgcgtat tggaggatca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc
     3901 accgattcta tgcccccact tctgagacaa gcaattgagc tgttcgacca tcagggagcc
     3961 gaacctgcct tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag
     4021 tgcgaaagcg gcgggccgga tatcggaggc ggatcaggtg gcggtagcgt ccccgatcgt
     4081 aaggctgcgg tctctcattg gcaacaggac gactataagg accacgacgg agactacaag
     4141 gatcatgata ttgattacaa agacgatgac gataaggcag gtaagtaatg cttttttttt
     4201 tctcgctctc atttttcaaa aacccacata aaaattgagg aaagggaaga attgttttct
     4261 ccttccagca cctcgtaatt tgacccgact gatggttccc attagtcaca taaagctgta
     4321 gtcaagtaca gacgtcccgc ggcttcgata ccgtcacgaa gcaacggatc cggccggcgc
     4381 ctagagaagg agtgagggct ggataaaggg aggatcgagg cggggtcgaa cgaggaggtt
     4441 caagggggag agacggggcg gatggaggaa gaggaggcgg aggcttaggg tgtacaaagg
     4501 gcttgaccca gggagggggg tcaaaagcca aggcttccca ggtcacgatg taggggacct
     4561 ggtctgggtg tccatgcggg ccaggtgaaa agaccttgat cttaacctgg gtgatgaggt
     4621 ctcggttaaa ggtgccgtct cgcggccatc cgacgttaaa ggttggccat tctgcagagc
     4681 agaaggtaac ccaacgtctc ttcttgacat ctaccgactg gttgtgagcg atccgctcga
     4741 catctttcca gtgacctaag gtcaaactta agggagtggt aacagtctgg ccctaatttt
     4801 cagacaaata cagaaacaca gtcagacaga gacaacacag aacgatgctg cagcgctgca
     4861 gcagacaaga cgcgcggctt cggttccaaa ccgaaagcaa aaattcagac ggaggcggga
     4921 actgttttag gttctcgtct cctaccagaa ccacatatcc tgacggggtc ggattccaca
     4981 tcgactccct tcctcaggtc gggccacaaa aacggccccc aaagtccctg ggacgtctcc
     5041 cagggttgcg gccgggtgtt cagaactcgt cagttccacc acgggtccgc cagatacaga
     5101 gctagttagc taactagtac agacgcaggc gcataacatc aaacatagac actagacaat
     5161 cggacagaca cagataagtt gctggccagc ttacctcccg gtggtgggtc ggtggtccct
     5221 gggcaggggt ctccaaatcc cggacgagcc cccaaatgaa agacccccgt cgtgggtagt
     5281 caatcactca gaggagaccc tcccaaggaa cagcgagacc acgattcgga tgcaaacagc
     5341 aagaggcttt attgggaata cgggtacccg ggcgacgcag tctatcggaa gactggcgcg
     5401 ccgagtgagg ggttgtgggc tcttttattg agctctgctt atatagacct cccaccgtac
     5461 acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg
     5521 ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc
     5581 ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt
     5641 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
     5701 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     5761 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
     5821 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
     5881 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
     5941 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt
     6001 caataatcaa tgtcaacatg gcggtaatgt tggacatgag ccaatataaa tgtacatatt
     6061 atgatatgga tacaacgtat gcaatggcca agctaattct tgaagacgaa agggcctcgt
     6121 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     6181 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6241 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     6301 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6361 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6421 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     6481 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6541 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6601 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6661 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6721 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6781 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6841 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6901 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6961 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     7021 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     7081 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     7141 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     7201 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     7261 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     7321 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     7381 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     7441 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     7501 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     7561 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     7621 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     7681 ct
//
