LOCUS       sequence-470322-        6486 bp    DNA     circular SYN 18-DEC-2025
DEFINITION  Retrovirus expressing mRFP1 with BBSI cloning sites for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6486)
  AUTHORS   Magnuson AM, Kiner E, Ergun A, Park JS, Asinovski N, Ortiz-Lopez A, 
            Kilcoyne A, Paoluzzi-Tomada E, Weissleder R, Mathis D, Benoist C
  TITLE     Identification and validation of a tumor-infiltrating Treg 
            transcriptional signature conserved across species and tumor types.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Nov 6;115(45):E10672-E10681. doi: 
            10.1073/pnas.1810580115. Epub 2018 Oct 22.
  PUBMED    30348759
REFERENCE   2  (bases 1 to 6486)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.1810580115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-11-6- 6"; volume: "115"; issue: "45"; pages: 
            "E10672-E10681"
COMMENT     Sequence Label: retro-gRNA-mRFP1
FEATURES             Location/Qualifiers
     source          1..6486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(476..1153)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVTKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(538..561)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     1005..1023
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1150..1159
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1178..1389)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(1451..1526)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1545..1793)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1603..1622)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1773..1793)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(1797..2213)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(1813..1829)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(1845..1867)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    2280..2621
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     LTR             2685..3201
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(3401..3420)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3548..3570)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3764..3783)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3883..3905
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3943..3961)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4029..4133
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4134..4202,4203..4994)
                     /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(4352..4371)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5165..5753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5654..5673
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5907..5924
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6041..6062
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6077..6082,6083..6100,6101..6107)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6115..6131
                     /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             join(6293..6486,1..321)
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 cgcgaacaga agcgagaagc gaactgattg gttagttcaa ataaggcaca gggtcatttc
       61 aggtccttgg ggcaccctgg aaacatctga tggttctcta gaaactgctg agggcgggac
      121 cgcatctggg gaccatctgt tcttggccct gagccggggc aggaactgct taccacagat
      181 atcctgtttg gcccatattc tgctgtctct ctgttcctaa ccttgatctg aacttctcta
      241 ttctcagtta tgtattttcc atgccttgca aaatggcgtt acttaagcta gcttgccaaa
      301 cctacaggtg gggtctttca ttcccccctt tttctggaga ctaaataaaa tcttttattt
      361 tatcgatatc agcgctttaa atttgcgcat gctagctata gttctagagg taccggttgt
      421 taacgttagc cggctacctg aagcttgcat gcctgcaggt cgactctaga gtcgcttagg
      481 cgccggtgga gtggcggccc tcggcgcgct cgtactgttc cacgatggtg tagtcctcgt
      541 tgtgggaggt gatgtccagc ttgatgtcgg tcttgtaggc gccgggcagc tgcacgggct
      601 tcttggccat gtaggtggtc ttgacctcgg cgtcgtagtg gccgccgtcc ttcagcttca
      661 gcctcatctt gatctcgccc ttcagggcgc cgtcctcggg gtacatccgc tcggtggagg
      721 cctcccagcc catggttttc ttctgcatta cggggccgtc ggaggggaag ttggtgccgc
      781 gcagcttcac cttgtagatg aactcgccgt cctgcaggga ggagtcctgg gtcacggtca
      841 ccacgccgcc gtcctcgaag ttcatcacgc gctcccactt gaagccctcg gggaaggaca
      901 gcttcaagta gtcggggatg tcggcggggt gcttcacgta ggccttggag ccgtactgga
      961 actgagggga caggatgtcc caggcgaagg gcagggggcc gcccttggtc accttcagct
     1021 tggcggtctg ggtgccctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt
     1081 ggccgttcac ggagccctcc atgcgcacct tgaagcgcat gaactccttg gtgacgtcct
     1141 cggaggaggc catggtggca gcgctctaga accggtcctg tgttctggcg gcaaacccgt
     1201 tgcgaaaaag aacgttcacg gcgactactg cacttatata cggttctccc ccaccctcgg
     1261 gaaaaaggcg gagccagtac acgacatcac tttcccagtt taccccgcgc caccttctct
     1321 aggcaccgga tcaattgccg acccctcccc ccaacttctc ggggactgtg ggcgatgtgc
     1381 gctctgccca ctgacgggca ccggagccaa ttcccactcc tttcaagacc tagctagcga
     1441 attcaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt
     1501 taacttgcta tttctagctc taaaacaggt cttctcgaag acccggtgtt tcgtcctttc
     1561 cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag
     1621 tccattttaa aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt
     1681 cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattctaat tatctctcta
     1741 acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctcgccggcg
     1801 cctagagaag gagtgagggc tggataaagg gaggatcgag gcggggtcga acgaggaggt
     1861 tcaaggggga gagacggggc ggatggagga agaggaggtg gaggcttagg gtgtacaaag
     1921 ggcttgaccc agggaggggg gtcaaaagcc aaggcttccc aggtcacgat gtaggggacc
     1981 tggtctgggt gtccatgcgg gccaggtgaa aagaccttga tcttaacctg ggtgatgagg
     2041 tctcggttaa aggtgccgtc tcgcggccat ccgacgttaa aggttggcca ttctgcagag
     2101 cagaaggtaa cccaacgtct cttcttgaca tctaccgact ggttgtgagc gatccgctcg
     2161 acatctttcc agtgacctaa ggtcaaactt aagggagtgg taacagtctg gccctaattt
     2221 tcagacaaat acagaaacac agtcagacag agacaacaca gaacgatgct gcagcgctgc
     2281 agcagacaag acgcgcggct tcggttccaa accgaaagca aaaattcaga cggaggcggg
     2341 aactgtttta ggttctcgtc tcctaccaga accacatatc ctgacggggt cggattccac
     2401 atcgactccc ttcctcaggt cgggccacaa aaacggcccc caaagtccct gggacgtctc
     2461 ccagggttgc ggccgggtgt tcagaactcg tcagttccac cacgggtccg ccagatacag
     2521 agctagttag ctaactagta cagacgcagg cgcaaacatt agatgccggc acaaacacgc
     2581 acaaagacag agacagacac gaaacgaccg ctggccagct tacctcccgg cgggggggtc
     2641 ggtggtccct gggcaggggt ctccaaatct cggtggaacc tccaaatgaa agacccccga
     2701 ggtgggcagt caatcaatct gaggagaccc tcccaaggat cagcgagtcc acgattcgga
     2761 tgcaaacagc aagaggcttt attgggaata cgggtacccg ggcgacgcag tctatcggag
     2821 gactggcgcg ccgagtgagg ggttgtgggc tcttttattg agctcgggga gcagaagcgc
     2881 gcgaacagaa gcgagaagcg aactgattgg ttagttcaaa taaggcacag ggtcatttca
     2941 ggtccttggg gcaccctgga aacatctgat ggttctctag aaactgctga gggcgggacc
     3001 gcatctgggg accatctgtt cttggccctg agccggggca ggaactgctt accacagata
     3061 tcctgtttgg cccatattct gctgtctctc tgttcctaac cttgatctga acttctctat
     3121 tctcagttat gtattttcca tgccttgcaa aatggcgtta cttaagctag cttgccaaac
     3181 ctacaggtgg ggtctttcat tccccccttt ttctggagac taaataaaat cttttatttt
     3241 atctatggct cgtactctat aggcttcagc tggtgatatt gttgagtcaa aactagagcc
     3301 tggaccactg atatcctgtc tttaacaaat tggactaatc gcctctacgc cggacgcatc
     3361 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     3421 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     3481 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     3541 gcgccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     3601 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     3661 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
     3721 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     3781 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3841 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3901 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     3961 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     4021 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4081 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4141 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4201 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4261 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4321 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4381 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4441 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4501 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4561 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4621 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4681 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4741 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4801 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4861 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4921 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4981 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5041 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5101 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5161 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5221 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5281 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5341 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5401 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5461 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5521 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5581 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5641 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5701 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5761 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5821 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5881 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5941 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     6001 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     6061 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     6121 gcggataaca atttcacaat tctccccagc tctttctgcg ctgtccataa tgaactattt
     6181 caggagtatt ggagtatgtg gattcaaaac agtggctaat tgagtcagga ttacttaata
     6241 ttcgactcct accctcagaa tgttgttctc caacttcaag aaactgttac ccatgaaaga
     6301 cccccgctga cgggtagtca atcactcaga ggagaccctc ccaaggaaca gcgagaccac
     6361 aagtcggatg caactgcaag agggtttatt ggatacacgg gtacccgggc gacgcagtct
     6421 atcggaggac tggcgcgccg agtgaggggt tgtgggctct tttattgagc tcggggagca
     6481 gaagcg
//
