LOCUS       sequence-486377-        7581 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Expresses a fluorescent shRNA against RB1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7581)
  AUTHORS   Shayler DWH, Stachelek K, Cambier L, Lee S, Bai J, Reid MW, 
            Weisenberger DJ, Bhat B, Aparicio JG, Kim Y, Singh M, Bay M, 
            Thornton ME, Doyle EK, Fouladian Z, Erberich SG, Grubbs BH, 
            Bonaguidi MA, Craft CM, Singh HP, Cobrinik D
  TITLE     Identification and characterization of early human photoreceptor 
            states and cell-state-specific retinoblastoma-related features.
  JOURNAL   bioRxiv [Preprint]. 2024 Aug 22:2023.02.28.530247. doi: 
            10.1101/2023.02.28.530247.
  PUBMED    38915659
REFERENCE   2  (bases 1 to 7581)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Aug 22:2023.02.28.530247. doi: 
            10.1101/2023.02.28.530247."
COMMENT     Sequence Label: pLKO.1C-YFP-shRB1-733
FEATURES             Location/Qualifiers
     source          1..7581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    241..358
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(455..703)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(513..532)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(683..703)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(1001..1045)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1230..1463
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    1956..2081
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             2128..2308
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(2323..2526)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2352..2372)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2527..2906
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(3007..3027)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3007..3025)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3046..3062)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3046..3062)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3059..3081)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3070..3086
                     /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(3094..3100,3101..3118,3119..3124))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3139..3160
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3277..3294)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3448..4036)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3528..3547)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4207..4998,4999..5067))
                     /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     4830..4849
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5068..5172)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5198..5653)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5335..5356)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5547..5566
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5780..5802
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5794..5811
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5795..5811
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5821..5840
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5821..5839
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      5860..5995
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6035..6156
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6046..6065)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6100..6119
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             6228..6461
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(join(6528..7241,7242..7244,7245..7247))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6573..6594)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6922..6941
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7181..7202
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      7244..7253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(join(7263..7581,1..192))
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(7326..7344)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
ORIGIN
        1 aggggcggag caggaagcgt cgccgggggg cccacaaggg tagcggcgaa gatccgggtg
       61 acgctgcgaa cggacgtgaa gaatgtgcga gacccagggt cggcgccgct gcgtttcccg
      121 gaaccacgcc cagagcagcc gcgtccctgc gcaaacccag ggctgccttg gaaaaggcgc
      181 aaccccaacc ccctcgagcc gcggccaaag tggatctctg ctgtccctgt aataaacccg
      241 aaaattttga atttttgtaa tttgtttttg taattcttta gtttgtatgt ctgttgctat
      301 tatgtctact attctttccc ctgcactgta ccccccaatc cccccttttc ttttaaaatt
      361 gtggatgaat actgccattt gtctcgaggt cgagaattca aaaaccacat tatttctagt
      421 ccaaactcga gtttggacta gaaataatgt ggccggtgtt tcgtcctttc cacaagatat
      481 ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag tccattttaa
      541 aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt cacgtatttt
      601 gtactaatat ctttgtgttt acagtcaaat taattccaat tatctctcta acagccttgt
      661 atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctcggtgaag ggggcggccg
      721 ctcgaggcta gtctcgtgat cgataccgtc gagatccgtt cactaatcga atggatctgt
      781 ctctgtctct ctctccacct tcttcttcta ttccttcggg cctgtcgggt cccctcgggg
      841 ttgggaggtg ggtctgaaac gataatggtg aatatccctg cctaactcta ttcactatag
      901 aaagtacagc aaaaactatt cttaaaccta ccaagcctcc tactatcatt atgaataatt
      961 ttatatacca cagccaattt gttatgttaa accaattcca caaacttgcc catttatcta
     1021 attccaataa ttcttgttca ttcttttctt gctggttttg cgattcttca attaaggagt
     1081 gtattaagct tgtgtaattg ttaatttctc tgtcccactc catccaggtc gtgtgattcc
     1141 aaatctgttc cagagattta ttactccaac tagcattcca aggcacagca gtggtgcaaa
     1201 tgagttttcc agagcaaccc caaatcccca ggagctgttg atcctttagg tatctttcca
     1261 cagccaggat tcttgcctgg agctgcttga tgccccagac tgtgagttgc aacagatgct
     1321 gttgcgcctc aatagccctc agcaaattgt tctgctgctg cactatacca gacaataatt
     1381 gtctggcctg taccgtcagc gtcattgacg ctgcgcccat agtgcttcct gctgctccca
     1441 agaacccaag gaacaaagct cctattccca ctgctctttt ttctctctgc accactcttc
     1501 tctttgcctt ggtgggtgct actcctaatg gttcaatttt tactacttta tatttatata
     1561 attcacttct ccaattgtcc ctcatatctc ctcctccagg tctgaagatc agcggccgct
     1621 tgctgtgcgg tggtcttact tttgttttgc tcttcctcta tcttgtctaa agcttccttg
     1681 gtgtctttta tctctatcct ttgatgcaca caatagaggg ttgctactgt attatataat
     1741 gatctaagtt cttctgatcc tgtctgaagg gatggttgta gctgtcccag tatttgtcta
     1801 cagccttctg atgtttctaa caggccagga ttaactgcga atcgttctag ctccctgctt
     1861 gcccatacta tatgttttaa tttatatttt ttctttcccc ctggccttaa ccgaattttt
     1921 tcccatcgcg atctaattct cccccgctta atactgacgc tctcgcaccc atctctctcc
     1981 ttctagcctc cgctagtcaa aatttttggc gtactcacca gtcgccgccc ctcgcctctt
     2041 gccgtgcgcg cttcagcaag ccgagtcctg cgtcgagaga gctcctctgg tttccctttc
     2101 gctttcaagt ccctgttcgg gcgccactgc tagagatttt ccacactgac taaaagggtc
     2161 tgagggatct ctagttacca gagtcacaca acagacgggc acacactact tgaagcactc
     2221 aaggcaagct ttattgaggc ttaagcagtg ggttccctag ttagccagag agctcccagg
     2281 ctcagatctg gtctaaccag agagaccccg gttcactaaa cgagctctgc ttatatagac
     2341 ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt
     2401 tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag
     2461 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg
     2521 catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca
     2581 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     2641 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     2701 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     2761 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     2821 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     2881 ttaataacta gtcaataatc aatgtcaaca tggcggtaat gttggacatg agccaatata
     2941 aatgtacata ttatgatatg gatacaacgt atgcaatggc caagcttgca gctccagctt
     3001 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     3061 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3121 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3181 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3241 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3301 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3361 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3421 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3481 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3541 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3601 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3661 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3721 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3781 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3841 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3901 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3961 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4021 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4081 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4141 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4201 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4261 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4321 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4381 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4441 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4501 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4561 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4621 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4681 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4741 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4801 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4861 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4921 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4981 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5041 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5101 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5161 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     5221 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     5281 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     5341 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     5401 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     5461 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     5521 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     5581 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     5641 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     5701 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     5761 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg
     5821 taatacgact cactataggg cgaattgggt acgtccctag gcctccaaaa aagcctcctc
     5881 actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa
     5941 ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg
     6001 gagttagggg cgggatagct agagccagac atgataagat acattgatga gtttggacaa
     6061 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     6121 ttatttgtaa ccattataag ctgcaataaa caagttcctc tcactctctg atattcattt
     6181 ctttgcaagt tataaatact gaataataag atgacatgaa ctactactgc tagagatttt
     6241 ccacactgac taaaagggtc tgagggatct ctagttacca gagtcacaca acagacgggc
     6301 acacactact tgaagcactc aaggcaagct ttattgaggc ttaagcagtg ggttccctag
     6361 ttagccagag agctcccagg ctcagatctg gtctaaccag agagacccag tacaagcaaa
     6421 aagcagatct tgtcttcgtt gggagtgaat tagcccttcc agtcccccct tttcttttaa
     6481 aaagtggcta agatctacag ctgccttgta agtcattggt cttaaagtta cttgtacagc
     6541 tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg
     6601 cgcttctcgt tggggtcttt gctcagggcg gactggtagc tcaggtagtg gttgtcgggc
     6661 agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc gagctgcacg
     6721 ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt cttctgcttg
     6781 tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg ccccaggatg
     6841 ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc
     6901 tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc
     6961 tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg
     7021 gggtagcggg cgaagcactg caggccgtag ccgaaggtgg tcacgagggt gggccagggc
     7081 acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg
     7141 ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc
     7201 aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatggt ggcgagatcc
     7261 ccctggggag agaggtcggt gattcggtca acgagggagc cgactgccga cgtgcgctcc
     7321 ggaggcttgc agaatgcgga acaccgcgcg ggcaggaaca gggcccacac taccgcccca
     7381 caccccgcct cccgcaccgc cccttcccgg ccgctgctct cggcgcgccc tgctgagcag
     7441 ccgctattgg ccacagccca tcgcggtcgg cgcgctgcca ttgctccctg gcgctgtccg
     7501 tctgcgaggg tactagtgag acgtgcggct tccgtttgtc acgtccggca cgccgcgaac
     7561 cgcaaggaac cttcccgact t
//
