LOCUS       sequence-200501-        4723 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A piggybac vector with 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 4723)
  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 4723)
  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
FEATURES             Location/Qualifiers
     source          1..4723
                     /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"
     repeat_region   803..837
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1328..1344)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1328..1344)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1341..1363)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1352..1368
                     /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(1376..1382,1383..1400,1401..1406))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1421..1442
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1559..1576)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1730..2318)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1810..1829)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2489..3280,3281..3349))
                     /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     3112..3131
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3350..3454)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3480..3935)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3617..3638)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3829..3848
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4062..4084
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4076..4093
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4077..4093
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   4223..4285
                     /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 ggcccattgg tatggctttt
      301 tccccgtatc cccccaggtg tctgcaggct caaagagcag cgagaagcgt tcagaggaaa
      361 gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg gctcggggat
      421 gcggggggag cgccggaccg gagcggagcc ccgggcggct cgctgctgcc ccctagcggg
      481 ggagggacgt aattacatcc ctgggggctt tggggggggg ctgtccctga tatctataac
      541 aagaaaatat atatataata agttatcacg taagtagaac atgaaataac aatataatta
      601 tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg
      661 cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag
      721 ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag
      781 agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta
      841 atctagctgc atcaggatca tatcgtcggg tcttttttcc ggctcagtca tcgcccaagc
      901 tggcgctatc tgggcatcgg ggaggaagaa gcccgtgcct tttcccgcga ggttgaagcg
      961 gcatggaaag agtttgccga ggatgactgc tgctgcattg acgttgagcg aaaacgcacg
     1021 tttaccatga tgattcggga aggtgtggcc atgcacgcct ttaacggtga actgttcgtt
     1081 caggccacct gggataccag ttcgtcgcgg cttttccgga cacagttccg gatggtcagc
     1141 ccgaagcgca tcagcaaccc gaacaatacc ggcgacagcc ggaactgccg tgccggtgtg
     1201 cagattaatg acagcggtgc ggcgctggga tattacgtca gcgaggacgg gtatcctggc
     1261 tggatgccgc agaaatggac atggataccc cgtgagttac ccggcgggcg cgcttggcgt
     1321 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     1381 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     1441 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     1501 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     1561 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1621 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     1681 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     1741 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     1801 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     1861 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1921 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1981 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2041 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2101 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2161 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2221 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     2281 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     2341 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     2401 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     2461 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     2521 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2581 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     2641 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     2701 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     2761 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     2821 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     2881 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2941 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3001 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3061 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3121 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     3181 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     3241 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     3301 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     3361 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     3421 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     3481 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     3541 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     3601 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     3661 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     3721 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     3781 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     3841 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     3901 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     3961 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     4021 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     4081 aacgacggcc agtgagcgcg cctcgttcat tcacgttttt gaacccgtgg aggacgggca
     4141 gactcgcggt gcaaatgtgt tttacagcgt gatggagcag atgaagatgc tcgacacgct
     4201 gcagaacacg cagctagatt aaccctagaa agataatcat attgtgacgt acgttaaaga
     4261 taatcatgtg taaaattgac gcatgtgttt tatcggtctg tatatcgagg tttatttatt
     4321 aatttgaata gatattaagt tttattatat ttacacttac atactaataa taaattcaac
     4381 aaacaattta tttatgttta tttatttatt aaaaaaaaca aaaactcaaa atttcttcta
     4441 taaagtaaca aaacttttat gagggacagc ccccccccaa agcccccagg gatgtaatta
     4501 cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc ggtccggcgc
     4561 tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca
     4621 cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac acctgggggg
     4681 atacggggaa aaggcctcca cggccactag tctagagcta gcc
//
