LOCUS       sequence-225514-        7201 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retrovirus expressing eGFP with BBSI cloning sites for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7201)
  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 7201)
  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"; 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-eGFP
FEATURES             Location/Qualifiers
     source          1..7201
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             123..639
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    703..1044
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1111..1527
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1457..1479
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1495..1511
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1531..1779
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1531..1551
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1702..1721
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        join(1797..1802,1803..1820,1821..1827)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1835..1851
                     /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)."
     primer_bind     1859..1875
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1859..1875
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1871..2443
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSSKLTLTKGNKSWSSTAVTAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFAGF
                     PRQALNRGLPLGFRFSALRHLDPKKLD"
     promoter        1896..1914
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    1927..2034
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     1951..1967
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1951..1967
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2001..2017)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2002..2018)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2042..2061)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2043..2061)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2068..2085)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2068..2084)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2313..2332)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     misc_RNA        2464..2539
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2601..2812
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2831..2840
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2837..2839,2840..2842,2843..3556)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2837..2839,2840..2842,2843..3553)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2882..2903)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3143..3162)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3490..3511
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3718..4232
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4394..4410
                     /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)."
     promoter        complement(join(4418..4424,4425..4442,4443..4448))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4463..4484
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4601..4618)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4772..5360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4852..4871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5531..6322,6323..6391))
                     /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     6154..6173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6392..6496)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6564..6582
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6620..6642)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6742..6761
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7105..7124
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aaagacagga tatcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag
       61 cctatagagt acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg
      121 ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag
      181 gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga acagagagac
      241 agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc
      301 aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat
      361 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca
      421 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca
      481 acccctcact cggcgcgcca gtcctccgat agactgcgtc gcccgggtac ccgtattccc
      541 aataaagcct cttgctgttt gcatccgaat cgtggactcg ctgatccttg ggagggtctc
      601 ctcagattga ttgactgccc acctcggggg tctttcattt ggaggttcca ccgagatttg
      661 gagacccctg cccagggacc accgaccccc ccgccgggag gtaagctggc cagcggtcgt
      721 ttcgtgtctg tctctgtctt tgtgcgtgtt tgtgccggca tctaatgttt gcgcctgcgt
      781 ctgtactagt tagctaacta gctctgtatc tggcggaccc gtggtggaac tgacgagttc
      841 tgaacacccg gccgcaaccc tgggagacgt cccagggact ttgggggccg tttttgtggc
      901 ccgacctgag gaagggagtc gatgtggaat ccgaccccgt caggatatgt ggttctggta
      961 ggagacgaga acctaaaaca gttcccgcct ccgtctgaat ttttgctttc ggtttggaac
     1021 cgaagccgcg cgtcttgtct gctgcagcgc tgcagcatcg ttctgtgttg tctctgtctg
     1081 actgtgtttc tgtatttgtc tgaaaattag ggccagactg ttaccactcc cttaagtttg
     1141 accttaggtc actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag
     1201 aagagacgtt gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg
     1261 cgagacggca cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct
     1321 ggcccgcatg gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt
     1381 gacccccctc cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca
     1441 tccgccccgt ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat
     1501 ccagccctca ctccttctct aggcgccggc gagggcctat ttcccatgat tccttcatat
     1561 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
     1621 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     1681 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     1741 ttcttggctt tatatatctt gtggaaagga cgaaacaccg ggtcttcacc ccaggcttta
     1801 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     1861 ggaaacagct atgaccatga ttacgccaag ctcgaaatta accctcacta aagggaacaa
     1921 aagctggagc tccaccgcgg tgacggccgc tctagaacta gtggatcccc cgggctgcag
     1981 gaattcgata tcaagcttat cgataccgtc gacctcgagg gggggcccgg tacccaattc
     2041 gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga
     2101 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     2161 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2221 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2281 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2341 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2401 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcagaaga
     2461 cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     2521 agtggcaccg agtcggtgct tttttgaatt cgctagctag gtcttgaaag gagtgggaat
     2581 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
     2641 gggaggggtc ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
     2701 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     2761 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggaccggtt
     2821 ctagagcgct gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2881 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2941 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     3001 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     3061 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     3121 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     3181 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     3241 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     3301 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     3361 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3421 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     3481 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3541 cgagctgtac aagtaaagcg gccgcgactc tagagtcgac ctgcaggcat gcaagcttca
     3601 ggtagccggc taacgttaac aaccggtacc tctagaacta tagctagcat gcgcaaattt
     3661 aaagcgctga tatcgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
     3721 aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg
     3781 aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag agacagcaga
     3841 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
     3901 agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc
     3961 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc
     4021 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct
     4081 cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtgt atccaataaa
     4141 ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag
     4201 tgattgacta cccgtcagcg ggggtctttc atgggtaaca gtttcttgaa gttggagaac
     4261 aacattctga gggtaggagt cgaatattaa gtaatcctga ctcaattagc cactgttttg
     4321 aatccacata ctccaatact cctgaaatag ttcattatgg acagcgcaga aagagctggg
     4381 gagaattgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4441 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4501 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4561 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4621 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4681 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4741 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4801 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4861 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4921 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4981 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5041 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5101 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5161 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5221 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5281 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5341 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5401 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5461 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5521 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5581 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5641 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5701 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5761 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5821 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5881 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5941 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6001 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6061 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6121 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6181 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6241 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6301 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6361 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6421 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6481 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     6541 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     6601 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     6661 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     6721 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     6781 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat
     6841 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     6901 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     6961 cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     7021 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     7081 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     7141 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggcgattag tccaatttgt
     7201 t
//
