LOCUS       sequence-256494-        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"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1992..2067
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2115..2232
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2281..2791
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2710..2728
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2813..3208
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             3245..3310
                     /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(3317..3319,3320..3322,3323..4039)
                     /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(3362..3383)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3623..3642)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3970..3991
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4120..4353
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4431..4552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4468..4487)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4522..4541
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4592..4727
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4747..4766)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4748..4766)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4776..4793)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4776..4792)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4785..4807)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4934..5389
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5021..5040)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5231..5252
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5415..5519
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5520..5588,5589..6380)
                     /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(5738..5757)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6551..7139
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7040..7059
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7293..7310
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7427..7448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7463..7468,7469..7486,7487..7493)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7501..7517
                     /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     7506..7528
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7525..7541
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7525..7541
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7560..7580
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7562..7580
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cgaccggttt
     1981 tttgcgtctc tgtttaagag ctagaaatag caagtttaaa taaggctagt ccgttatcaa
     2041 cttgaaaaag tggcaccgag tcggtgcttt tttgaattct agatcttgag acaaatggca
     2101 gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     2161 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     2221 attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag
     2281 ggggcccggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc
     2341 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc
     2401 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc
     2461 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac
     2521 ggaagccgca cgtcttacta gtaccctcgc agacggacag cgccagggag caatggcagc
     2581 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag
     2641 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct
     2701 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct
     2761 cgttgaccga atcaccgacc tctctcccca gggggatcca ccggagctta ccatggccaa
     2821 gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat
     2881 ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt
     2941 cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg
     3001 cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa
     3061 caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg
     3121 gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt
     3181 gctgccctct ggttatgtgt gggagggcca gggatctggc gaagccagac acaaacagaa
     3241 aattgtgaaa cagactttga attttgacct tctcaagttg gcgggagacg tggagtccaa
     3301 cccagggccc gctagcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     3361 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     3421 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     3481 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     3541 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     3601 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     3661 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     3721 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     3781 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     3841 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3901 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     3961 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     4021 cgagctgtac aagtcctaaa cgcgttaagt cgactttaag accaatgact tacaaggcag
     4081 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     4141 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     4201 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     4261 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     4321 gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta
     4381 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     4441 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4501 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4561 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4621 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4681 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
     4741 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     4801 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     4861 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     4921 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4981 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     5041 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5101 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5161 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5221 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5281 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5341 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5401 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5461 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5521 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5581 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5641 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5701 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5761 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5821 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5881 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5941 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6001 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6061 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6121 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6181 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6241 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6301 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6361 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6421 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6481 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6541 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6601 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6661 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6721 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6781 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6841 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6901 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6961 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     7021 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     7081 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7141 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     7201 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     7261 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     7321 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     7381 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     7441 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     7501 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
     7561 caattaaccc tcactaaagg gaacaaaagc tggagctgca agc
//
