LOCUS       sequence-441234-        6991 bp    DNA     circular SYN 15-APR-2025
DEFINITION  CRISPR-screening.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6991)
  AUTHORS   Michlits G, Hubmann M, Wu SH, Vainorius G, Budusan E, Zhuk S, 
            Burkard TR, Novatchkova M, Aichinger M, Lu Y, Reece-Hoyes J, Nitsch 
            R, Schramek D, Hoepfner D, Elling U
  TITLE     CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 
            screens.
  JOURNAL   Nat Methods. 2017 Dec;14(12):1191-1197. doi: 10.1038/nmeth.4466. 
            Epub 2017 Oct 16.
  PUBMED    29039415
REFERENCE   2  (bases 1 to 6991)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nmeth.4466"; journalName: "Nat Methods"; date: "2017-12"; 
            volume: "14"; issue: "12"; pages: "1191-1197"
COMMENT     Sequence Label: pRetro-UMI
FEATURES             Location/Qualifiers
     source          1..6991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(550..1341,1342..1410))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1173..1192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1411..1515)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1583..1601
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        1701..2080
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2081..2284
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2235..2255
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2285..2460
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    2523..2722
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             2923..3338
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     3268..3290
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     3306..3322
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        3429..3677
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3429..3449
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3600..3619
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3982..4481
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4017..4040)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4414..4433
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4495..5298
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4558..4577)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5168..5187
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             5469..5894
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5997..6019)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6155..6175)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6160..6489
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6340..6475
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6511..6530
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6611..6628)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6782..6991,1..379))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6862..6881)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
       61 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
      121 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
      181 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
      241 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
      301 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
      361 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
      421 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      481 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
      541 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
      601 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
      661 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
      721 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
      781 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
      841 tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat
      901 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      961 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     1021 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     1081 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     1141 accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt
     1201 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     1261 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     1321 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1381 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     1441 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1501 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     1561 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtg ttcaagaatt
     1621 agcttggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg
     1681 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac
     1741 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     1801 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     1861 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     1921 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     1981 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     2041 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     2101 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     2161 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     2221 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     2281 agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt
     2341 cgcccgggta cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggtctc
     2401 gctgttcctt gggagggtct cctctgagtg attgactacc cacgacgggg gtctttcatt
     2461 tgggggctcg tccgggattt ggagacccct gcccagggac caccgaccca ccaccgggag
     2521 gtaagctggc cagcaactta tctgtgtctg tccgattgtc tagtgtctat gtttgatgtt
     2581 atgcgcctgc gtctgtacta gttagctaac tagctctgta tctggcggac ccgtggtgga
     2641 actgacgagt tctgaacacc cggccgcaac cctgggagac gtcccaggga ctttgggggc
     2701 cgtttttgtg gcccgacctg aggaagggag tcgatgtgga atccgacccc gtcaggatat
     2761 gtggttctgg taggagacga gaacctaaaa cagttcccgc ctccgtctga atttttgctt
     2821 tcggtttgga accgaagccg cgcgtcttgt ctgctgcagc gctgcagcat cgttctgtgt
     2881 tgtctctgtc tgactgtgtt tctgtatttg tctgaaaatt agggccagac tgttaccact
     2941 cccttaagtt tgaccttagg tcactggaaa gatgtcgagc ggatcgctca caaccagtcg
     3001 gtagatgtca agaagagacg ttggttacct tctgctctgc agaatggcca acctttaacg
     3061 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     3121 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     3181 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     3241 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     3301 cctcccttta tccagccctc actccttctc taggcgccgg aattgaagat ctccgcggtt
     3361 aattaaaatg atacggcgac caccgagatc tgtcttgatc taggagctac tcatcaacgg
     3421 tcacatgtga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     3481 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     3541 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     3601 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     3661 ggaaaggacg aaacaccggg tcttcgctcg agagaagacc tgtttaagag ctatgctgga
     3721 aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     3781 tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt
     3841 agcgcgtgcg ccaattctgc agacaaatgg ctctagaggt acccgttaag atcggaagag
     3901 cacacgtctg aactccagtc acaaaattat ctcgtatgcc gtcttatgct tgttaattaa
     3961 caattgacgc gtaattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
     4021 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
     4081 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     4141 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     4201 cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc
     4261 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     4321 gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg
     4381 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     4441 ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcagc caatatggga
     4501 tcggccattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     4561 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     4621 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     4681 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     4741 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4801 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4861 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4921 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4981 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     5041 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     5101 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     5161 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     5221 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     5281 cttgacgagt tcttctgagg ggatccgtcg acctgcagcc aagcttatcg ataccgtcga
     5341 gatttaatta agatatcatc agtggtccag gctctagttt tgactcaaca atatcaccag
     5401 ctgaagccta tagagtacga gccatagata aaataaaaga ttttatttag tctccagaaa
     5461 aaggggggaa tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt
     5521 tgcaaggcat ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca
     5581 gatggaacag ggtcgaccct agagaaccat cagatgtttc cagggtgccc caaggacctg
     5641 aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc
     5701 gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct
     5761 ccgattgact gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc
     5821 gacttgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc
     5881 gggggtcttt catttggggg ctcgtccggg atcgggagac ccctgcccag ggaccaccga
     5941 cccaccaccg ggaggtaagc tggctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     6001 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     6061 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     6121 tcacgtagcg atagcggagt gtagatccgg ctgtggaatg tgtgtcagtt agggtgtgga
     6181 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     6241 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     6301 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     6361 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     6421 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     6481 ttttgcaaaa agcttactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     6541 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     6601 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     6661 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     6721 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6781 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6841 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6901 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6961 gcgtggcgct ttctcatagc tcacgctgta g
//
