LOCUS       Exported                4131 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Piggybac transposon sgRNA cloning backbone with MS2 loops at 
            tetraloop and stemloop 2. Contains BbsI sites for insertion of 
            spacer sequences..
ACCESSION   .
VERSION     .
KEYWORDS    PB-sgRNA(MS2)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4131)
  AUTHORS   Li S, Zhang A, Xue H, Li D, Liu Y
  TITLE     One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of 
            Multiple Genes.
  JOURNAL   Mol Ther Nucleic Acids. 2017 Sep 15;8:64-76. doi: 
            10.1016/j.omtn.2017.06.007. Epub 2017 Jun 15.
  PUBMED    28918057
REFERENCE   2  (bases 1 to 4131)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4131
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     173..191
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(229..251)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     351..370
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        451..469
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     451..468
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    535..556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        571..601
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    609..625
                     /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     614..636
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     633..649
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     633..649
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        670..688
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   871..933
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1227..1467
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1227..1247
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1398..1417
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1511..1529
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        1581..1599
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     repeat_region   2077..2111
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2235..2254)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2236..2254)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2341..2358)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2512..3100)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2592..2611)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3271..4131)
                     /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     3894..3913
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
      181 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
      241 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      301 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      361 gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta
      421 atacataacc ttatgtatca tacacatacg atttaggtga cactatagaa ctcgacctcg
      481 aggctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      541 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      601 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      661 caagctcgaa attaaccctc actaaaggga acaaaagctg gagctcgtct ttgatcaaaa
      721 cgcaaatcga cgaaaatgtg tcggacaata tcaagtcgat gagcgaaaaa ctaaaaaggc
      781 tagaatacga caatctcaca gacagcgttg agatatacgg tattcacgac agcaggctga
      841 ataataaaaa aattagaaac tattatttaa ccctagaaag ataatcatat tgtgacgtac
      901 gttaaagata atcatgcgta aaattgacgc atgtgtttta tcgatctgta tatcgaggtt
      961 tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata
     1021 aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa
     1081 tttcttctat aaagtaacaa aacttttaaa cattctctct tttacaaaaa taaacttatt
     1141 ttgtacttta aaaacagtca tgttgtatta taaaataagt aattagctta acttatacat
     1201 aatagaaaca aattatactt attaatgagg gcctatttcc catgattcct tcatatttgc
     1261 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     1321 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     1381 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     1441 tggctttata tatcttgtgg aaaggacgaa acaccgggtc ttcgagaaga cctgttttag
     1501 agctaggcca acatgaggat cacccatgtc tgcagggcct agcaagttaa aataaggcta
     1561 gtccgttatc aacttggcca acatgaggat cacccatgtc tgcagggcca agtggcaccg
     1621 agtcggtgct ttttttgaat tctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     1681 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcatt taaatccatc
     1741 gaagcagctc cagcctacac aatcgctcaa gacgtgtaat gcttttatta tatattagtc
     1801 acgatatcta taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa
     1861 taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt
     1921 cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc
     1981 acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg
     2041 cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta
     2101 tctttctagg gttaaaaaag atttgcgctt tactcgacct aaactttaaa cacgtcatag
     2161 aatcttcgtt tgacaaaaac cacattgtgg ggtaccgagc tcgaattcat cgatgatatc
     2221 agatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg ttaacctgca
     2281 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     2341 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2401 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2461 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2521 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2581 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2641 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2701 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2761 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2821 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2881 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2941 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3001 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3061 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3121 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3181 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3241 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3301 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3361 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3421 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3481 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3541 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3601 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3661 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3721 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3781 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3841 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3901 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3961 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4021 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4081 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca t
//
