LOCUS       sequence-261410-        7603 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 promoter expresses customizable Spyo-guide; PGK promoter 
            expresses blasticidin resistance and 2A site provides EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7603)
  AUTHORS   DeWeirdt PC, Sangree AK, Hanna RE, Sanson KR, Hegde M, Strand C, 
            Persky NS, Doench JG
  TITLE     Genetic screens in isogenic mammalian cell lines without single cell
            cloning.
  JOURNAL   Nat Commun. 2020 Feb 6;11(1):752. doi: 10.1038/s41467-020-14620-6.
  PUBMED    32029722
REFERENCE   2  (bases 1 to 7603)
  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: "Nat 
            Commun."; date: "2020-02-6"; volume: "11(1):752. doi"; pages: " 
            10.1038/s41467-020-14620-6"
COMMENT     Sequence Label: pRDA_186
FEATURES             Location/Qualifiers
     source          1..7603
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             259..303
                     /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"
     promoter        481..729
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     481..501
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     652..671
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        756..831
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    879..996
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1045..1555
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1474..1492
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1577..1972
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             2009..2074
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(2081..2083,2084..2086,2087..2803)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKS"
     primer_bind     complement(2126..2147)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2387..2406)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2734..2755
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             2884..3117
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3195..3316
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3232..3251)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3286..3305
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3356..3491
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3511..3530)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3512..3530)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3540..3557)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3540..3556)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3549..3571)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3698..4153
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3785..3804)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3995..4016
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4179..4283
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4284..4352,4353..5144)
                     /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(4502..4521)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5315..5903
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5804..5823
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6057..6074
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6191..6212
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6227..6232,6233..6250,6251..6257)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6265..6281
                     /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     6270..6292
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6289..6305
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6289..6305
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6324..6344
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6326..6344
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6372..6598
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6599..6779
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6826..6951
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    join(7444..7603,1..74)
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
ORIGIN
        1 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
       61 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
      121 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
      181 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
      241 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
      301 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
      361 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
      421 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccaga
      481 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
      541 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      601 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      661 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      721 cgaaacaccg gagacgaccg gttttttgcg tctctgttta agagctagaa atagcaagtt
      781 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgaa
      841 ttctagatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
      901 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
      961 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     1021 gatccacttt ggcgccggct cgagggggcc cgggttgcgc cttttccaag gcagccctgg
     1081 gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg gcgccgaccc
     1141 tgggtctcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct
     1201 tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt
     1261 cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctt actagtaccc tcgcagacgg
     1321 acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca atagcggctg
     1381 ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg
     1441 ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca agcctccgga
     1501 gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc cccaggggga
     1561 tccaccggag cttaccatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag
     1621 agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc
     1681 tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc
     1741 ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg
     1801 tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca
     1861 ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc
     1921 gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg gccagggatc
     1981 tggcgaagcc agacacaaac agaaaattgt gaaacagact ttgaattttg accttctcaa
     2041 gttggcggga gacgtggagt ccaacccagg gcccgctagc atggtgagca agggcgagga
     2101 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     2161 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     2221 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2281 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2341 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2401 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2461 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     2521 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     2581 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     2641 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     2701 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2761 cgccgggatc actctcggca tggacgagct gtacaagtcc taaacgcgtt aagtcgactt
     2821 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     2881 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     2941 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     3001 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     3061 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     3121 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     3181 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3241 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3301 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
     3361 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3421 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     3481 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     3541 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     3601 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     3661 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     3721 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3781 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3841 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3901 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3961 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4021 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     4081 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4141 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4201 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4261 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     4321 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4381 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4441 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     4501 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     4561 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4621 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4681 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4741 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4801 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4861 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4921 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4981 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     5041 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     5101 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     5161 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5221 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5281 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5341 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5401 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5461 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5521 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5581 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5641 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5701 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5761 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5821 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5881 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5941 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6001 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     6061 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     6121 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     6181 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     6241 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     6301 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
     6361 tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag
     6421 ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag
     6481 gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac
     6541 cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg
     6601 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     6661 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     6721 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     6781 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
     6841 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
     6901 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
     6961 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     7021 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     7081 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     7141 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     7201 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     7261 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     7321 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     7381 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     7441 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     7501 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     7561 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctc
//
