LOCUS       sequence-458099-        6462 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PiggyBac transposon vector backbone for cloning of sgRNA compatible 
            with S.pyogenes dCas9 enzyme. BbsI Golden Gate site upstream of the 
            scaffold for protospacer insertion. Puromycin selection marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6462)
  TITLE     PiggyBac CRISPR-dCas9 fusion protein and gRNA donor plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6462)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: PB_gRNA_S.pyogenes_scaffold_BB
FEATURES             Location/Qualifiers
     source          1..6462
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1162..1617)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1299..1320)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1511..1530
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1744..1766
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1758..1775
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1759..1775
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1905..1967
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2406..2654
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2406..2426
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2577..2596
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2823..3034
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             3047..3315
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             3342..3941
                     /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(3342..3361)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3838..3858
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4001..4021)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4063..4184
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4100..4119)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4154..4173
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4947..4981
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5472..5488)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5472..5488)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5485..5507)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5496..5512
                     /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(5520..5526,5527..5544,5545..5550))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5565..5586
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5703..5720)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5874..6462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5954..5973)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
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
//
