LOCUS       sequence-234705-        7977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows Gateway cloning of gene of interest into retroviral pMSCV 
            vector (with Puromycin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7977)
  AUTHORS   Benitez-Guijarro M, Lopez-Ruiz C, Tarnauskaite Z, Murina O, Mian 
            Mohammad M, Williams TC, Fluteau A, Sanchez L, Vilar-Astasio R, 
            Garcia-Canadas M, Cano D, Kempen MH, Sanchez-Pozo A, Heras SR, 
            Jackson AP, Reijns MA, Garcia-Perez JL
  TITLE     RNase H2, mutated in Aicardi-Goutieres syndrome, promotes LINE-1 
            retrotransposition.
  JOURNAL   EMBO J. 2018 Aug 1;37(15). pii: embj.201798506. doi: 
            10.15252/embj.201798506. Epub 2018 Jun 29.
  PUBMED    29959219
REFERENCE   2  (bases 1 to 7977)
  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: "EMBO J."; 
            date: "2018-08-1"; volume: "37(15). pii: embj.201798506. doi"; 
            pages: " 10.15252/embj.201798506. Epub 2018 Jun 29"
COMMENT     Sequence Label: pMSCVpuro-DEST
FEATURES             Location/Qualifiers
     source          1..7977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     377..400
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    453..577
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(618..923)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(720..739)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1265..1924)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1839..1858
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1978..1984,1985..2002,2003..2008))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2033..2157)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2183..2599)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(2199..2215)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(2231..2253)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    2666..3007
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     LTR             3071..3587
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(3787..3806)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3934..3956)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4150..4169)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4269..4291
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4329..4347)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4415..4519
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4520..4588,4589..5380)
                     /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     complement(4738..4757)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5551..6139
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6040..6059
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6293..6310
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6427..6448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6463..6468,6469..6486,6487..6493)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6501..6517
                     /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)."
     LTR             6679..7193
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     CDS             complement(7293..7892)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7376..7396)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     7873..7892
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(join(7913..7977,1..435))
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(join(7961..7977,1..3))
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
ORIGIN
        1 atgccgggcc tccggaggac cttcgggcgc ccgccccgcc cctgagcccg cccctgagcc
       61 cgcccccgga cccacccctt cccagcctct gagcccagaa agcgaaggag caaagctgct
      121 attggccgct gccccaaagg cctacccgct tccattgctc agcggtgctg tccatctgca
      181 cgagactagt gagacgtgct acttccattt gtcacgtcct gcacgacgcg agctgcgggg
      241 cgggggggaa cttcctgact aggggaggag tagaaggtgg cgcgaagggg ccaccaaaga
      301 acggagccgg ttggcgccta ccggtggatg tggaatgtgt gcgaggccag aggccacttg
      361 tgtagcgcca agtgcccagc ggggctgcta aagcgcatgc tccagactgc cttgggaaaa
      421 gcgcctcccc tacccggtag aattcgttat caaccacttt gtacaagaaa gctgaacgag
      481 aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta
      541 cataatactg taaaacacaa catatccagt cactatggtc gacctgcaga ctggctgtgt
      601 ataagggagc ctgacattta tattccccag aacatcaggt taatggcgtt tttgatgtca
      661 ttttcgcggt ggctgagatc agccacttct tccccgataa cggagaccgg cacactggcc
      721 atatcggtgg tcatcatgcg ccagctttca tccccgatat gcaccaccgg gtaaagttca
      781 cgggagactt tatctgacag cagacgtgca ctggccaggg ggatcaccat ccgtcgcccg
      841 ggcgtgtcaa taatatcact ctgtacatcc acaaacagac gataacggct ctctctttta
      901 taggtgtaaa ccttaaactg catttcacca gcccctgttc tcgtcagcaa aagagccgtt
      961 catttcaata aaccgggcga cctcagccat cccttcctga ttttccgctt tccagcgttc
     1021 ggcacgcaga cgacgggctt cattctgcat ggttgtgctt accagaccgg agatattgac
     1081 atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa ctgtcactgt aatacgctgc
     1141 ttcatagcat acctcttttt gacatacttc gggtatacat atcagtatat attcttatac
     1201 cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc ttttagtaag ccggatccag
     1261 atctttacgc cccgccctgc cactcatcgc agtactgttg taattcatta agcattctgc
     1321 cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc atcagcacct
     1381 tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat
     1441 tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca
     1501 tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt
     1561 gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa
     1621 acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca
     1681 gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg cgggcaagaa
     1741 tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg
     1801 taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa
     1861 aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg atttttttct
     1921 ccattttagc ttccttagct cctgaaaatc tcgacggatc ctaactcaaa atccacacat
     1981 tatacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgcggcc gccatagtga
     2041 ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa
     2101 tatattgata tttatatcat tttacgtttc tcgttcagct tttttgtaca aacttgttga
     2161 taacctcgag agatctaatt ccggcgccta gagaaggagt gagggctgga taaagggagg
     2221 atcgaggcgg ggtcgaacga ggaggttcaa gggggagaga cggggcggat ggaggaagag
     2281 gaggcggagg cttagggtgt acaaagggct tgacccaggg aggggggtca aaagccaagg
     2341 cttcccaggt cacgatgtag gggacctggt ctgggtgtcc atgcgggcca ggtgaaaaga
     2401 ccttgatctt aacctgggtg atgaggtctc ggttaaaggt gccgtctcgc ggccatccga
     2461 cgttaaaggt tggccattct gcagagcaga aggtaaccca acgtctcttc ttgacatcta
     2521 ccgactggtt gtgagcgatc cgctcgacat ctttccagtg acctaaggtc aaacttaagg
     2581 gagtggtaac agtctggccc taattttcag acaaatacag aaacacagtc agacagagac
     2641 aacacagaac gatgctgcag cgctgcagca gacaagacgc gcggcttcgg ttccaaaccg
     2701 aaagcaaaaa ttcagacgga ggcgggaact gttttaggtt ctcgtctcct accagaacca
     2761 catatcctga cggggtcgga ttccacatcg actcccttcc tcaggtcggg ccacaaaaac
     2821 ggcccccaaa gtccctggga cgtctcccag ggttgcggcc gggtgttcag aactcgtcag
     2881 ttccaccacg ggtccgccag atacagagct agttagctaa ctagtacaga cgcaggcgca
     2941 aacattagat gccggcacaa acacgcacaa agacagagac agacacgaaa cgaccgctgg
     3001 ccagcttacc tcccggcggg ggggtcggtg gtccctgggc aggggtctcc aaatctcggt
     3061 ggaacctcca aatgaaagac ccccgaggtg ggcagtcaat caatctgagg agaccctccc
     3121 aaggatcagc gagtccacga ttcggatgca aacagcaaga ggctttattg ggaatacggg
     3181 tacccgggcg acgcagtcta tcggaggact ggcgcgccga gtgaggggtt gtgggctctt
     3241 ttattgagct cggggagcag aagcgcgcga acagaagcga gaagcgaact gattggttag
     3301 ttcaaataag gcacagggtc atttcaggtc cttggggcac cctggaaaca tctgatggtt
     3361 ctctagaaac tgctgagggc gggaccgcat ctggggacca tctgttcttg gccctgagcc
     3421 ggggcaggaa ctgcttacca cagatatcct gtttggccca tattctgctg tctctctgtt
     3481 cctaaccttg atctgaactt ctctattctc agttatgtat tttccatgcc ttgcaaaatg
     3541 gcgttactta agctagcttg ccaaacctac aggtggggtc tttcattccc ccctttttct
     3601 ggagactaaa taaaatcttt tattttatct atggctcgta ctctataggc ttcagctggt
     3661 gatattgttg agtcaaaact agagcctgga ccactgatat cctgtcttta acaaattgga
     3721 ctaatcgcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
     3781 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     3841 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     3901 ccatctcctt gcatgcacca ttccttgcgc cgtcgtttta caacgtcgtg actgggaaaa
     3961 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     4021 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     4081 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     4141 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4201 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4261 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4321 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4381 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4441 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4501 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4561 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4621 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4681 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4741 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4801 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4861 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     4921 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     4981 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5041 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5101 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5161 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5221 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5281 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5341 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5401 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5461 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5521 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5581 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5641 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5701 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5761 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5821 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5881 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5941 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6001 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6061 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6121 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6181 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6241 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     6301 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     6361 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     6421 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     6481 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacaattctc cccagctctt
     6541 tctgcgctgt ccataatgaa ctatttcagg agtattggag tatgtggatt caaaacagtg
     6601 gctaattgag tcaggattac ttaatattcg actcctaccc tcagaatgtt gttctccaac
     6661 ttcaagaaac tgttacccat gaaagacccc cgctgacggg tagtcaatca ctcagaggag
     6721 accctcccaa ggaacagcga gaccacaagt cggatgcaac tgcaagaggg tttattggat
     6781 acacgggtac ccgggcgacg cagtctatcg gaggactggc gcgccgagtg aggggttgtg
     6841 ggctctttta ttgagctcgg ggagcagaag cgcgcgaaca gaagcgagaa gcgaactgat
     6901 tggttagttc aaataaggca cagggtcatt tcaggtcctt ggggcaccct ggaaacatct
     6961 gatggttctc tagaaactgc tgagggcggg accgcatctg gggaccatct gttcttggcc
     7021 ctgagccggg gcaggaactg cttaccacag atatcctgtt tggcccatat tctgctgtct
     7081 ctctgttcct aaccttgatc tgaacttctc tattctcagt tatgtatttt ccatgccttg
     7141 caaaatggcg ttacttaagc tagcttgcca aacctacagg tggggtcttt cattcccccc
     7201 tttttctgga gactaaataa aatcttttat tttatcgatg catggggtcg tgcgctcctt
     7261 tcggtcgggc gctgcgggtc gtggggcggg cgtcaggcac cgggcttgcg ggtcatgcac
     7321 caggtgcgcg gtccttcggg cacctcgacg tcggcggtga cggtgaagcc gagccgctcg
     7381 tagaagggga ggttgcgggg cgcggaggtc tccaggaagg cgggcacccc ggcgcgctcg
     7441 gccgcctcca ctccggggag cacgacggcg ctgcccagac ccttgccctg gtggtcgggc
     7501 gagacgccga cggtggccag gaaccacgcg ggctccttgg gccggtgcgg cgccaggagg
     7561 ccttccatct gttgctgcgc ggccagccgg gaaccgctca actcggccat gcgcgggccg
     7621 atctcggcga acaccgcccc cgcttcgacg ctctccggcg tggtccagac cgccaccgcg
     7681 gcgccgtcgt ccgcgaccca caccttgccg atgtcgagcc cgacgcgcgt gaggaagagt
     7741 tcttgcagct cggtgacccg ctcgatgtgg cggtccggat cgacggtgtg gcgcgtggcg
     7801 gggtagtcgg cgaacgcggc ggcgagggtg cgtacggccc tggggacgtc gtcgcgggtg
     7861 gcgaggcgca ccgtgggctt gtactcggtc atggtaagct tgggctgcag gtcgaaaggc
     7921 ccggagatga ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc gtgcaga
//
