LOCUS       sequence-69518-e        7750 bp    DNA     circular SYN 14-APR-2025
DEFINITION  sgRNA expression on PiggyBac vector with Neo-selectable marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7750)
  AUTHORS   Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan 
            S, Shivalila CS, Dadon DB, Jaenisch R
  TITLE     Multiplexed activation of endogenous genes by CRISPR-on, an 
            RNA-guided transcriptional activator system.
  JOURNAL   Cell Res. 2013 Aug 27. doi: 10.1038/cr.2013.122.
  PUBMED    23979020
REFERENCE   2  (bases 1 to 7750)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Res. 
            2013 Aug 27. doi: 10.1038/cr.2013.122."
COMMENT     Sequence Label: pAC103-PBNeo-U6-sgRNA-Expression
FEATURES             Location/Qualifiers
     source          1..7750
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   299..333
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(649..672)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1046..1065
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1127..1930
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1952..1969)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1958..2165
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2778..3026
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2778..2798
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2949..2968
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3045..3120
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   4022..4084
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4122..4141)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4123..4141)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4148..4165)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4148..4164)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4157..4179)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             4302..4604
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4483..4502
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4953..5747
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4953..5747
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(join(5997..6788,6789..6857))
                     /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     6620..6639
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6981..7569
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7470..7489
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7723..7740
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca
      241 gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttttaacc
      301 ctagaaagat agtctgcgta aaattgacgc atgcattctt gaaatattgc tctctctttc
      361 taaatagcgc gaatccgtcg ctgtgcattt aggacatctc agtcgccgct tggagctccc
      421 gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt gaactataac gaccgcgtga
      481 gtcaaaatga cgcatgatta tcttttacgt gacttttaag atttaactca tacgataatt
      541 atattgttat ttcatgttct acttacgtga taacttatta tatatatatt ttcttgttat
      601 agatatcaac tagaatgcta gccagtactt ttcccaaggc agtctggagc atgcgcttta
      661 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
      721 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
      781 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
      841 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
      901 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
      961 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     1021 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     1081 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gccaatatgg gatcggccat
     1141 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     1201 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     1261 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     1321 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     1381 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     1441 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     1501 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     1561 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     1621 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     1681 tgatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     1741 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     1801 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     1861 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     1921 gttcttctga gctctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     1981 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2041 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2101 gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct
     2161 ggggaggcgc gttggatccc cgggtaccga gttgggagct cacggggaca gccccccccc
     2221 aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg
     2281 gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag
     2341 cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt
     2401 gagcctgcag acacctgggg ggatacgggg aaaaagcttt aggctgaaag agagatttag
     2461 aatgacagtc tagtgggagc tcacggggac agcccccccc caaagccccc agggatgtaa
     2521 ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg
     2581 cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg
     2641 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg
     2701 gggatacggg gaaaaagctt taggctgaaa gagagattta gaatgacaga actcgatttc
     2761 attgcagact ggcgcgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2821 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     2881 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2941 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     3001 atatcttgtg gaaaggacga aacaccgggt cttcgagaag acctgtttta gagctagaaa
     3061 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     3121 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt
     3181 gcgccaattc tgcagacaaa tggcacgcgc ctggatcccc gggtaccgag ttgggagctc
     3241 acggggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg
     3301 ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc
     3361 cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga
     3421 acgcttctcg ctgctctttg agcctgcaga cacctggggg gatacgggga aaaagcttta
     3481 ggctgaaaga gagatttaga atgacagtct agtgggagct cacggggaca gccccccccc
     3541 aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg
     3601 gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag
     3661 cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt
     3721 gagcctgcag acacctgggg ggatacgggg aaaaagcttt aggctgaaag agagatttag
     3781 aatgacagaa ctcgatttca ttgcagactg gccggccact agtacgcgcc ggctcgacat
     3841 actagttaaa agttttgtta ctttatagaa gaaattttga gtttttgttt ttttttaata
     3901 aataaataaa cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat
     3961 aataaaactt aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac
     4021 acatgcgtca attttacgca tgattatctt taacgtacgt cacaatatga ttatctttct
     4081 agggttaaaa gggcgaattc gcggccgcta aattcaattc gccctatagt gagtcgtatt
     4141 acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     4201 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     4261 ccgatcgccc ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag gtttacacct
     4321 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc
     4381 cggggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc
     4441 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata
     4501 tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa
     4561 atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca ggcatgagat
     4621 tatcaaaaag gatcttcacc tagatccttt tcacgtagaa agccagtccg cagaaacggt
     4681 gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa
     4741 agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat
     4801 ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct
     4861 gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagct
     4921 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     4981 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     5041 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     5101 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     5161 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     5221 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     5281 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     5341 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     5401 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     5461 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     5521 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     5581 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     5641 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     5701 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaatt attaacgctt
     5761 acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatca
     5821 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5881 tcaaatatgt atccgctcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5941 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6001 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6061 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6121 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6181 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6241 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6301 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6361 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6421 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6481 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6541 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6601 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6661 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6721 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6781 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6841 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6901 tgtctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6961 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     7021 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     7081 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     7141 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     7201 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     7261 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     7321 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7381 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7441 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7501 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7561 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7621 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     7681 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     7741 gaagcggaag
//
