ORFtag_LambdaN-SD_Frame2

LOCUS       ORFtag_LambdaN-SD_Frame2       7128 bp    DNA     circular SYN 14-APR-2025
DEFINITION  ORFtag viral retroviral vector containing a constitutively active 
            promoter and a LambdaN 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 7128)
  TITLE     Proteome-scale tagging and functional screening in mammalian cells 
            by ORFtag
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7128)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: ORFtag_LambdaN-SD_Frame2
FEATURES             Location/Qualifiers
     source          1..7128
                     /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..4337,4338..4340,4341..4349)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3930..3947)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4157..4176
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      4356..4365
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4362..4427
                     /codon_start=1
                     /product="N-terminal RNA-binding domain from the lambda 
                     bacteriophage antiterminator protein N (Baron-Benhamou et 
                     al., 2004)"
                     /label=lambda N peptide
                     /translation="MDAQTRRRERRAEKQAQWKAAN"
     CDS             4488..4523
                     /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             4527..4592
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(4792..5208)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(4808..4824)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(4840..4862)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    5409..5608
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             5671..5846
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     promoter        complement(5847..6050)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5876..5896)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6051..6430
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(6530..6548)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6616..6720
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6721..6789,6790..7128,1..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(6939..6958)
                     /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 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     3841 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     3901 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     3961 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
     4021 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     4081 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     4141 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     4201 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt
     4261 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     4321 aaaaacacga tgataatatc gccacaacca gatctgccac catggacgca caaacacgac
     4381 gacgtgagcg tcgcgctgag aaacaagctc aatggaaagc tgcaaacggt ggatctcctc
     4441 caagcttgat ggctgatatc ggaggcggat caggtggcgg tagcgtcccc gatcgtaagg
     4501 ctgcggtctc tcattggcaa caggacgact ataaggacca cgacggagac tacaaggatc
     4561 atgatattga ttacaaagac gatgacgata aggcaggtaa gtaatgcttt tttttttctc
     4621 gctctcattt ttcaaaaacc cacataaaaa ttgaggaaag ggaagaattg ttttctcctt
     4681 ccagcacctc gtaatttgac ccgactgatg gttcccatta gtcacataaa gctgtagtca
     4741 agtacagacg tcccgcggct tcgataccgt cacgaagcaa cggatccggc cggcgcctag
     4801 agaaggagtg agggctggat aaagggagga tcgaggcggg gtcgaacgag gaggttcaag
     4861 ggggagagac ggggcggatg gaggaagagg aggcggaggc ttagggtgta caaagggctt
     4921 gacccaggga ggggggtcaa aagccaaggc ttcccaggtc acgatgtagg ggacctggtc
     4981 tgggtgtcca tgcgggccag gtgaaaagac cttgatctta acctgggtga tgaggtctcg
     5041 gttaaaggtg ccgtctcgcg gccatccgac gttaaaggtt ggccattctg cagagcagaa
     5101 ggtaacccaa cgtctcttct tgacatctac cgactggttg tgagcgatcc gctcgacatc
     5161 tttccagtga cctaaggtca aacttaaggg agtggtaaca gtctggccct aattttcaga
     5221 caaatacaga aacacagtca gacagagaca acacagaacg atgctgcagc gctgcagcag
     5281 acaagacgcg cggcttcggt tccaaaccga aagcaaaaat tcagacggag gcgggaactg
     5341 ttttaggttc tcgtctccta ccagaaccac atatcctgac ggggtcggat tccacatcga
     5401 ctcccttcct caggtcgggc cacaaaaacg gcccccaaag tccctgggac gtctcccagg
     5461 gttgcggccg ggtgttcaga actcgtcagt tccaccacgg gtccgccaga tacagagcta
     5521 gttagctaac tagtacagac gcaggcgcat aacatcaaac atagacacta gacaatcgga
     5581 cagacacaga taagttgctg gccagcttac ctcccggtgg tgggtcggtg gtccctgggc
     5641 aggggtctcc aaatcccgga cgagccccca aatgaaagac ccccgtcgtg ggtagtcaat
     5701 cactcagagg agaccctccc aaggaacagc gagaccacga ttcggatgca aacagcaaga
     5761 ggctttattg ggaatacggg tacccgggcg acgcagtcta tcggaagact ggcgcgccga
     5821 gtgaggggtt gtgggctctt ttattgagct ctgcttatat agacctccca ccgtacacgc
     5881 ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga
     5941 ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt
     6001 gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata
     6061 gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg
     6121 gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata
     6181 tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt
     6241 caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg
     6301 cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac
     6361 tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat
     6421 aatcaatgtc aacatggcgg taatgttgga catgagccaa tataaatgta catattatga
     6481 tatggataca acgtatgcaa tggccaagct aattcttgaa gacgaaaggg cctcgtgata
     6541 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     6601 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     6661 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     6721 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     6781 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     6841 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     6901 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     6961 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     7021 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     7081 tgcagtgctg ccataaccat gagtgataac actgcggcca acttactt
//