LOCUS       sequence-409039-        8348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for cloning single guides (Chen tracrRNA PMID: 24360272) or
            guide libraries with untranscribed barcodes and a tracrRNA of 
            choice.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8348)
  AUTHORS   Schmierer B, Botla SK, Zhang J, Turunen M, Kivioja T, Taipale J
  TITLE     CRISPR/Cas9 screening using unique molecular identifiers.
  JOURNAL   Mol Syst Biol. 2017 Oct 9;13(10):945. doi: 10.15252/msb.20177834.
  PUBMED    28993443
REFERENCE   2  (bases 1 to 8348)
  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: "Mol Syst 
            Biol."; date: "2017-10-9"; volume: "13(10):945. doi"; pages: " 
            10.15252/msb.20177834"
COMMENT     Sequence Label: lentiGuide-Puro-Chen-tracrRNA_2xBsmBI
FEATURES             Location/Qualifiers
     source          1..8348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    78..199
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(115..134)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     169..188
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      239..374
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(394..413)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(395..413)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(423..440)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(423..439)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(432..454)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      581..1036
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(668..687)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     878..899
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1062..1166
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1167..1235,1236..2027)
                     /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(1385..1404)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2198..2786
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2687..2706
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2940..2957
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3074..3095
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3110..3115,3116..3133,3134..3140)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3148..3164
                     /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     3153..3175
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3172..3188
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3172..3188
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3207..3227
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3209..3227
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3255..3481
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             3482..3662
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3709..3834
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4327..4560
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4745..4789
                     /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        4967..5215
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4967..4987
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5138..5157
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    5393..5510
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5649..6827
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5880..6818
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6775..6795
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             6840..7439
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6840..6859)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7336..7356
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7455..8043
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7508..7528)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7926..7937)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8115..8348
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
       61 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
      121 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
      181 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
      241 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
      301 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
      361 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
      421 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
      481 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
      541 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
      601 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
      661 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
      721 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
      781 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
      841 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
      901 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
      961 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     1021 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1081 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1141 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1201 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1261 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     1321 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1381 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     1441 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1501 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1561 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1621 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1681 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     1741 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1801 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     1861 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     1921 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     1981 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2041 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2101 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2161 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2221 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2281 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2341 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2401 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2461 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2521 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2581 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2641 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2701 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2761 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2821 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2881 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2941 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     3001 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     3061 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     3121 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     3181 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
     3241 agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat
     3301 gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt
     3361 aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg
     3421 aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa
     3481 cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     3541 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     3601 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     3661 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     3721 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     3781 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     3841 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     3901 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     3961 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     4021 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     4081 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     4141 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     4201 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     4261 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     4321 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     4381 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     4441 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     4501 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     4561 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     4621 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     4681 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     4741 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     4801 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     4861 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     4921 accattatcg tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc
     4981 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa
     5041 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     5101 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     5161 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga
     5221 cggttgttaa ttaatgtacg tctctgttta agagctatgc tggaaacagc atagcaagtt
     5281 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgaa
     5341 ttcaagcttg gcgtaactag atcttgagac aaatggcagt attcatccac aattttaaaa
     5401 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     5461 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     5521 acagggacag cagagatcca ctttggcgcc ggctcgaggg ggcccgggtg caaagatgga
     5581 taaagtttta aacagagagg aatctttgca gctaatggac cttctaggtc ttgaaaggag
     5641 tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
     5701 agttgggggg aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact
     5761 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
     5821 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
     5881 taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc
     5941 cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa
     6001 gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg
     6061 aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc
     6121 tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt
     6181 ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt
     6241 ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg
     6301 gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc
     6361 tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt
     6421 cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa
     6481 aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg
     6541 cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc
     6601 acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt
     6661 atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca gcttggcact
     6721 tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc
     6781 ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgacgt acggccacca
     6841 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     6901 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     6961 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     7021 tcggcaaggt gtgggtcgcg gacgacggcg ccgccgtggc ggtctggacc acgccggaga
     7081 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     7141 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     7201 ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca
     7261 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     7321 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     7381 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa
     7441 cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     7501 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     7561 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     7621 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     7681 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     7741 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     7801 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     7861 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     7921 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     7981 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     8041 cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     8101 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     8161 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     8221 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     8281 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     8341 ctctagca
//
