LOCUS       sequence-437540-        7680 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 
            Frame1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7680)
  TITLE     Proteome-scale tagging and functional screening in mammalian cells 
            by ORFtag
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7680)
  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            "Unpublished"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_Frame1
FEATURES             Location/Qualifiers
     source          1..7680
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      624..1212
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1113..1132
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1366..1383
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(1464..1483)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(1505..1834)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1519..1654
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1819..1839
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     1975..1997
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2451..2950
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2486..2509)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2883..2902
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2963..3757
                     /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(3017..3036)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3627..3646
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    join(3763..4336,4337..4339,4340..4348)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3929..3946)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4156..4175
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4364..4384
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5042..5077
                     /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             5081..5146
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(5344..5760)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(5360..5376)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(5392..5414)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    5961..6160
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             6223..6398
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     promoter        complement(6399..6602)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6428..6448)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6603..6982
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(7082..7100)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7168..7272
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7273..7341,7342..7680,1..453)
                     /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(7491..7510)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
       61 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
      121 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
      181 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
      241 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
      301 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
      361 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
      421 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
      481 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
      541 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
      601 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
      661 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      721 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
      781 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      841 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      901 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      961 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     1021 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     1081 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     1141 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     1201 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     1261 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     1321 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     1381 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     1441 caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtaa
     1501 gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag
     1561 aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tggggcggag
     1621 aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc gggactatgg
     1681 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     1741 ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     1801 agcctgggga ctttccacac cctaactgac acacattcca cagccggatc tacactccgc
     1861 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     1921 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     1981 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ccagcttacc
     2041 tcccggtggt gggtcggtgg tccctgggca ggggtctccc gatcccggac gagcccccaa
     2101 atgaaagacc cccgctgacg ggtagtcaat cactcagagg agaccctccc aaggaacagc
     2161 gagaccacaa gtcggatgca actgcaagag ggtttattgg atacacgggt acccgggcga
     2221 ctcagtcaat cggaggactg gcgccccgag tgaggggttg tgctagcttg ccaaacctac
     2281 aggtggggtc tttcattccc ccctttttct ggagactaaa taaaatcttt tattttatcg
     2341 atggtcaagt tccttctggt tctggctctg ctcatcgtcc tgcaggaccg gacgtgactg
     2401 gagttcagac gtgtgctctt ccgatctcat gttaactcga gaattctacc gggtagggga
     2461 ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg
     2521 ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc
     2581 tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc
     2641 ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag
     2701 tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg
     2761 ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg
     2821 gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc
     2881 ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc
     2941 gggcctttcg gaattcgcca ccatgattga acaagatgga ttgcacgcag gttctccggc
     3001 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3061 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3121 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     3181 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3241 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3301 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3361 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3421 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     3481 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     3541 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3601 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3661 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     3721 catcgccttc tatcgccttc ttgacgagtt cttctgacgc gtcccctctc cctccccccc
     3781 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     3841 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     3901 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     3961 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     4021 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     4081 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     4141 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     4201 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     4261 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     4321 aaaacacgat gataatatcg ccacaaccag atctgccacc atgcccaaaa agaagaggaa
     4381 agtgatgtct agactggaca agagcaaagt cataaactct gctctggaat tactcaatga
     4441 agtcggtatc gaaggcctga cgacaaggaa actcgctcaa aagctgggag ttgagcagcc
     4501 taccctgtac tggcacgtga agaacaagcg ggccctgctc gatgccctgg caatcgagat
     4561 gctggacagg catcataccc acttctgccc cctggaaggc gagtcatggc aagactttct
     4621 gcggaacaac gccaagtcat tccgctgtgc tctcctctca catcgcgacg gggctaaagt
     4681 gcatctcggc acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt
     4741 cctgtgtcag caaggcttct ccctggagaa cgcactgtac gctctgtccg ccgtgggcca
     4801 ctttacactg ggctgcgtat tggaggatca ggagcatcaa gtagcaaaag aggaaagaga
     4861 gacacctacc accgattcta tgcccccact tctgagacaa gcaattgagc tgttcgacca
     4921 tcagggagcc gaacctgcct tccttttcgg cctggaacta atcatatgtg gcctggagaa
     4981 acagctaaag tgcgaaagcg gcgggccgga tatcggaggc ggatcaggtg gcggtagcgt
     5041 ccccgatcgt aaggctgcgg tctctcattg gcaacaggac gactataagg accacgacgg
     5101 agactacaag gatcatgata ttgattacaa agacgatgac gataagcagg taagtaatgc
     5161 tttttttttc tcgctctcat ttttcaaaaa cccacataaa aattgaggaa agggaagaat
     5221 tgttttctcc ttccagcacc tcgtaatttg acccgactga tggttcccat tagtcacata
     5281 aagctgtagt caagtacaga cgtcccgcgg cttcgatacc gtcacgaagc aacggatccg
     5341 gccggcgcct agagaaggag tgagggctgg ataaagggag gatcgaggcg gggtcgaacg
     5401 aggaggttca agggggagag acggggcgga tggaggaaga ggaggcggag gcttagggtg
     5461 tacaaagggc ttgacccagg gaggggggtc aaaagccaag gcttcccagg tcacgatgta
     5521 ggggacctgg tctgggtgtc catgcgggcc aggtgaaaag accttgatct taacctgggt
     5581 gatgaggtct cggttaaagg tgccgtctcg cggccatccg acgttaaagg ttggccattc
     5641 tgcagagcag aaggtaaccc aacgtctctt cttgacatct accgactggt tgtgagcgat
     5701 ccgctcgaca tctttccagt gacctaaggt caaacttaag ggagtggtaa cagtctggcc
     5761 ctaattttca gacaaataca gaaacacagt cagacagaga caacacagaa cgatgctgca
     5821 gcgctgcagc agacaagacg cgcggcttcg gttccaaacc gaaagcaaaa attcagacgg
     5881 aggcgggaac tgttttaggt tctcgtctcc taccagaacc acatatcctg acggggtcgg
     5941 attccacatc gactcccttc ctcaggtcgg gccacaaaaa cggcccccaa agtccctggg
     6001 acgtctccca gggttgcggc cgggtgttca gaactcgtca gttccaccac gggtccgcca
     6061 gatacagagc tagttagcta actagtacag acgcaggcgc ataacatcaa acatagacac
     6121 tagacaatcg gacagacaca gataagttgc tggccagctt acctcccggt ggtgggtcgg
     6181 tggtccctgg gcaggggtct ccaaatcccg gacgagcccc caaatgaaag acccccgtcg
     6241 tgggtagtca atcactcaga ggagaccctc ccaaggaaca gcgagaccac gattcggatg
     6301 caaacagcaa gaggctttat tgggaatacg ggtacccggg cgacgcagtc tatcggaaga
     6361 ctggcgcgcc gagtgagggg ttgtgggctc ttttattgag ctctgcttat atagacctcc
     6421 caccgtacac gcctaccgcc catttgcgtc aatggggcgg agttgttacg acattttgga
     6481 aagtcccgtt gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt
     6541 ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc
     6601 accatggtaa tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag
     6661 gtcatgtact gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc
     6721 gtacttggca tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc
     6781 acccattgac gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt
     6841 gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat
     6901 gtaacgcgga actccatata tgggctatga actaatgacc ccgtaattga ttactattaa
     6961 taactagtca ataatcaatg tcaacatggc ggtaatgttg gacatgagcc aatataaatg
     7021 tacatattat gatatggata caacgtatgc aatggccaag ctaattcttg aagacgaaag
     7081 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     7141 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     7201 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     7261 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     7321 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     7381 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     7441 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     7501 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     7561 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     7621 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
//
