LOCUS       bGG298_PB_gRNA_S.pyogen 6462 bp ds-DNA     circular     29-AUG-2024
DEFINITION  A piggybac vector with puromycin resistance and a U6 promoter that
            allows cloning of guide RNAs.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
COMMENT     Imported using the Genbank importer. File name:
            addgene-plasmid-104537-sequence-200502.gbk
FEATURES             Location/Qualifiers
     CDS             complement(171..1031)
                     /label="AmpR"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="confers resistance to ampicillin, carbenicillin, andrelated antibiotics"
                     /gene="bla"
                     /product="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     promoter        complement(1032..1136)
                     /label="AmpR promoter"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /gene="bla"
     rep_origin      complement(1162..1617)
                     /label="f1 ori"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
                     /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
                     /direction="LEFT"
     repeat_region   1905..1967
                     /label="piggyBac right (3') inverted repeat"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)"
     promoter        2406..2646
                     /label="U6 promoter"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2535..2557
                     /label="Foward Primer in U6 Promoter"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
                     /note="forward primer in U6 promoter used in QC for sgRNA protospacer cloning, primer ID: GG2349"
     misc_feature    2655..2677
                     /label="cut out"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
                     /note="digest with BbsI to remove this cut out region, replace with custom protospacer sequence for final sgRNA"
     protein_bind    2657..2662
                     /label="BbsI (BpiI) recognition site"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /note="BbsI restriction site part of Golden Gate cloning site"
     primer_bind     complement(2666..2688)
                     /label="Reverse Primer in backbone cut out region"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="Reverse primer in backbone cut out region, used in QC for sgRNA protospacer cloning. When used with forward primer in U6 promoter, should produce no PCR product if new protospacer correctly cloned in. Primer ID: GG2350"
     protein_bind    2670..2675
                     /label="BbsI (BpiI recognition site)"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="BbsI restriction site part of Golden Gate cloning site"
     misc_feature    2678..2770
                     /label="S. Pyogenes Scaffold"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     primer_bind     complement(2735..2754)
                     /label="Reverse Primer in Scaffold sequence"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
                     /note="Reverse primer in scaffold region, used in QC for sgRNA protospacer cloning. When used with forward primer in U6 promoter, should produce PCR product, use as positive control. Primer ID: GG2351"
     promoter        2823..3034
                     /label="EF-1-alpha core promoter"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="core promoter for human elongation factor EF-1-alpha"
     LTR             3047..3315
                     /label="5' LTR (truncated)"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1"
     CDS             3342..3941
                     /label="PuroR"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="confers resistance to puromycin"
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
     polyA_signal    4063..4184
                     /label="SV40 poly(A) signal"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="SV40 polyadenylation signal"
     repeat_region   4947..4981
                     /label="piggyBac left (5') inverted repeat"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
                     /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)"
     protein_bind    5496..5512
                     /label="lac operator"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /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)."
                     /bound_moiety="lac repressor encoded by lacI"
     promoter        complement(5520..5550)
                     /label="lac promoter"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
                     /note="promoter for the E. coli lac operon"
     protein_bind    5565..5586
                     /label="CAP binding site"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="CAP binding activates transcription in the presence of cAMP."
                     /bound_moiety="E. coli catabolite activator protein"
     rep_origin      complement(5874..6462)
                     /label="ori"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
                     /direction="LEFT"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     1201 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     1261 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     1321 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     1381 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     1441 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     1501 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     1561 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     1621 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     1681 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     1741 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcctcgttc attcacgttt
     1801 ttgaacccgt ggaggacggg cagactcgcg gtgcaaatgt gttttacagc gtgatggagc
     1861 agatgaagat gctcgacacg ctgcagaaca cgcagctaga ttaaccctag aaagataatc
     1921 atattgtgac gtacgttaaa gataatcatg tgtaaaattg acgcatgtgt tttatcggtc
     1981 tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt
     2041 acatactaat aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa
     2101 caaaaactca aaatttcttc tataaagtaa caaaactttt atgagggaca gccccccccc
     2161 aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg
     2221 gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag
     2281 cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt
     2341 gagcctgcag acacctgggg ggatacgggg aaaaggcctc cacggccact agtctagagc
     2401 tagccgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     2461 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     2521 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     2581 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     2641 aaggacgaaa caccgggtct tcATTAATGg aagacACgtt taagagctaa gctggaaaca
     2701 gcatagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     2761 tgcttttttt aattcgggcc cgcggccgcg aaggatctgc gatcgctccg gtgcccgtca
     2821 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
     2881 aacgggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
     2941 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
     3001 tctttttcgc aacgggtttg ccgccagaac acagctgaag cttcgagggg ctcgcatctc
     3061 tccttcacgc gcccgccgcc ctacctgagg ccgccatcca cgccggttga gtcgcgttct
     3121 gccgcctccc gcctgtggtg cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct
     3181 caggtcgaga ccgggccttt gtccggcgct cccttggagc ctacctagac tcagccggct
     3241 ctccacgctt tgcctgaccc tgcttgctca actctacgtc tttgtttcgt tttctgttct
     3301 gcgccgttac agatccaagc tgtgaccggc gcctacgcta gatgaccgag tacaagccca
     3361 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     3421 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     3481 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     3541 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     3601 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     3661 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     3721 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     3781 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     3841 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     3901 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg agtcgacaat caacctctgg
     3961 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     4021 gtggatacgc tgctttaatg cctttgtatc atgcgttaac taaacttgtt tattgcagct
     4081 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4141 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggaattga
     4201 ctcaaatgat gtcaattagt ctatcagaag ctcatctggt ctcccttccg ggggacaaga
     4261 catccctgtt taatatttaa acagcagtgt tcccaaactg ggttcttata tcccttgctc
     4321 tggtcaacca ggttgcaggg tttcctgtcc tcacaggaac gaagtcccta aagaaacagt
     4381 ggcagccagg tttagccccg gaattgactg gattcctttt ttagggccca ttggtatggc
     4441 tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag
     4501 gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg
     4561 ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag
     4621 cgggggaggg acgtaattac atccctgggg gctttggggg ggggctgtcc ctgatatcta
     4681 taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa taacaatata
     4741 attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt cattttgact
     4801 cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg
     4861 ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag
     4921 aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg
     4981 gttaatctag ctgcatcagg atcatatcgt cgggtctttt ttccggctca gtcatcgccc
     5041 aagctggcgc tatctgggca tcggggagga agaagcccgt gccttttccc gcgaggttga
     5101 agcggcatgg aaagagtttg ccgaggatga ctgctgctgc attgacgttg agcgaaaacg
     5161 cacgtttacc atgatgattc gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt
     5221 cgttcaggcc acctgggata ccagttcgtc gcggcttttc cggacacagt tccggatggt
     5281 cagcccgaag cgcatcagca acccgaacaa taccggcgac agccggaact gccgtgccgg
     5341 tgtgcagatt aatgacagcg gtgcggcgct gggatattac gtcagcgagg acgggtatcc
     5401 tggctggatg ccgcagaaat ggacatggat accccgtgag ttacccggcg ggcgcgcttg
     5461 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5521 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5581 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5641 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5701 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5761 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5821 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5881 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5941 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6001 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6061 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6121 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6181 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6241 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6301 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6361 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6421 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aa
//