LOCUS       sequence-200502-        6371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A piggybac vector with puromycin resistance and a U6 promoter that 
            allows cloning of guide RNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6371)
  AUTHORS   Kalhor R, Kalhor K, Mejia L, Leeper K, Graveline A, Mali P, Church 
            GM
  TITLE     Developmental barcoding of whole mouse via homing CRISPR.
  JOURNAL   Science. 2018 Aug 9. pii: science.aat9804. doi: 
            10.1126/science.aat9804.
  PUBMED    30093604
REFERENCE   2  (bases 1 to 6371)
  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: "Science. 
            2018 Aug 9. pii: science.aat9804. doi: 10.1126/science.aat9804."
COMMENT     Sequence Label: PB-U6insert-EF1puro
FEATURES             Location/Qualifiers
     source          1..6371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        327..538
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             551..819
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             846..1445
                     /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(846..865)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1342..1362
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1505..1525)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    1567..1688
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1604..1623)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1658..1677
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   2451..2485
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2976..2992)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2976..2992)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2989..3011)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3000..3016
                     /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(3024..3030,3031..3048,3049..3054))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3069..3090
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3207..3224)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3378..3966)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3458..3477)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4137..4928,4929..4997))
                     /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     4760..4779
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4998..5102)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5128..5583)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5265..5286)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5477..5496
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5710..5732
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5724..5741
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5725..5741
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   5871..5933
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gtccccggat ccgatttaaa ttcgaattcg ggcccgcggc cgcgaaggat
      301 ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
      361 gttgggggga ggggtcggca attgaacggg tgcctagaga aggtggcgcg gggtaaactg
      421 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
      481 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagct
      541 gaagcttcga ggggctcgca tctctccttc acgcgcccgc cgccctacct gaggccgcca
      601 tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt ggtgcctcct gaactgcgtc
      661 cgccgtctag gtaagtttaa agctcaggtc gagaccgggc ctttgtccgg cgctcccttg
      721 gagcctacct agactcagcc ggctctccac gctttgcctg accctgcttg ctcaactcta
      781 cgtctttgtt tcgttttctg ttctgcgccg ttacagatcc aagctgtgac cggcgcctac
      841 gctagatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
      901 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
      961 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     1021 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     1081 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     1141 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     1201 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     1261 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     1321 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     1381 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     1441 cctgagtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     1501 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcgt
     1561 taactaaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     1621 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     1681 gtatcttatc atgtctggaa ttgactcaaa tgatgtcaat tagtctatca gaagctcatc
     1741 tggtctccct tccgggggac aagacatccc tgtttaatat ttaaacagca gtgttcccaa
     1801 actgggttct tatatccctt gctctggtca accaggttgc agggtttcct gtcctcacag
     1861 gaacgaagtc cctaaagaaa cagtggcagc caggtttagc cccggaattg actggattcc
     1921 ttttttaggg cccattggta tggctttttc cccgtatccc cccaggtgtc tgcaggctca
     1981 aagagcagcg agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc
     2041 tgtccccgca cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc
     2101 gggcggctcg ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg
     2161 ggggggggct gtccctgata tctataacaa gaaaatatat atataataag ttatcacgta
     2221 agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa
     2281 agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta
     2341 ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca
     2401 cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa
     2461 ttttacgcag actatctttc tagggttaat ctagctgcat caggatcata tcgtcgggtc
     2521 ttttttccgg ctcagtcatc gcccaagctg gcgctatctg ggcatcgggg aggaagaagc
     2581 ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag tttgccgagg atgactgctg
     2641 ctgcattgac gttgagcgaa aacgcacgtt taccatgatg attcgggaag gtgtggccat
     2701 gcacgccttt aacggtgaac tgttcgttca ggccacctgg gataccagtt cgtcgcggct
     2761 tttccggaca cagttccgga tggtcagccc gaagcgcatc agcaacccga acaataccgg
     2821 cgacagccgg aactgccgtg ccggtgtgca gattaatgac agcggtgcgg cgctgggata
     2881 ttacgtcagc gaggacgggt atcctggctg gatgccgcag aaatggacat ggataccccg
     2941 tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3001 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3061 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3121 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3181 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3241 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3301 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3361 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3421 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3481 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3541 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3601 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3661 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3721 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3781 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3841 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3901 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3961 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4021 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4081 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4141 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4201 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4261 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4321 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4381 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4441 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4501 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4561 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4621 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4681 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4741 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4801 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4861 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4921 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4981 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5041 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5101 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     5161 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     5221 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     5281 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     5341 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     5401 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     5461 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     5521 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     5581 cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     5641 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     5701 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcc tcgttcattc
     5761 acgtttttga acccgtggag gacgggcaga ctcgcggtgc aaatgtgttt tacagcgtga
     5821 tggagcagat gaagatgctc gacacgctgc agaacacgca gctagattaa ccctagaaag
     5881 ataatcatat tgtgacgtac gttaaagata atcatgtgta aaattgacgc atgtgtttta
     5941 tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt
     6001 acacttacat actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa
     6061 aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa acttttatga gggacagccc
     6121 ccccccaaag cccccaggga tgtaattacg tccctccccc gctagggggc agcagcgagc
     6181 cgcccggggc tccgctccgg tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg
     6241 ggacagcccg ggcacgggga aggtggcacg ggatcgcttt cctctgaacg cttctcgctg
     6301 ctctttgagc ctgcagacac ctggggggat acggggaaaa ggcctccacg gccactagtc
     6361 tagagctagc c
//
