LOCUS       sequence-335409-        7992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2 transposon vector of empty U6-S.aureus Cas9 gRNA cassette and 
            CAG-driven Cre-NeoR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7992)
  AUTHORS   Biegler MT, Fedrigo O, Collier P, Mountcastle J, Haase B, Tilgner 
            HU, Jarvis ED
  TITLE     Induction of an immortalized songbird cell line allows for gene 
            characterization and knockout by CRISPR-Cas9.
  JOURNAL   Sci Rep. 2022 Mar 14;12(1):4369. doi: 10.1038/s41598-022-07434-7.
  PUBMED    35288582
REFERENCE   2  (bases 1 to 7992)
  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: "Sci Rep."; 
            date: "2022-03-14"; volume: "12(1):4369. doi"; pages: " 
            10.1038/s41598-022-07434-7"
COMMENT     Sequence Label: 
            pMB1052:miniTol2-U6-(empty)SaCas9_gRNA-CAG-Cre-P2A-NeoR-WPRE
FEATURES             Location/Qualifiers
     source          1..7992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..18
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2..18
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     25..44
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        25..43
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(56..67,68..255)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     promoter        298..546
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     298..318
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     469..488
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        567..642
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        712..1015
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1293..2309
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2233..2255
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2317..2336
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2367..2387
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3429..3485
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3492..4286
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3546..3565)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4156..4175
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4372..4391)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4437..4492
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4491..4510)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     misc_feature    join(4933..5095,5096..5107)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        complement(5158..5176)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5159..5176)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5194..5210)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5194..5210)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5207..5229)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5218..5234
                     /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(5242..5248,5249..5266,5267..5272))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5287..5308
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5425..5442)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5596..6184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5676..5695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6355..7146,7147..7215))
                     /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     6978..6997
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7216..7320)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7398..7853)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7535..7556)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7747..7766
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(7979..7992,1..9)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt gggcccagag
       61 gtgtaaagta cttgagtaat tttacttgat tactgtactt aagtattatt tttggggatt
      121 tttactttac ttgagtacaa ttaaaaatca atacttttac ttttacttaa ttacattttt
      181 ttagaaaaaa aagtactttt tactccttac aattttattt acagtcaaaa agtacttatt
      241 ttttggagat cacttcattc tattttccct aagattgaat tgcgctgatg cccagatgag
      301 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
      361 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
      421 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
      481 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
      541 aacaccggag accacggcag gtctcagttt tagtactctg gaaacagaat ctactaaaac
      601 aaggcaaaat gccgtgttta tctcgtcaac ttgttggcga gattttttta ctagttatta
      661 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      721 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      781 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      841 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      901 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      961 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga
     1021 ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt
     1081 gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg
     1141 cgccrggggg gggggggggg gggggggggg gggggggggs grggsggaga ggkgcggcgg
     1201 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
     1261 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
     1321 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
     1381 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat
     1441 gacggcttgt ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt
     1501 tgtgcggggg gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt
     1561 gcggctccgc gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc
     1621 tccgcagtgt gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct
     1681 gcgaggggaa caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg
     1741 gcgcgtcggt cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc
     1801 cggcttcggg tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg
     1861 ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg
     1921 gggaggggcg cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc
     1981 attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg
     2041 cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt
     2101 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc
     2161 cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac
     2221 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc
     2281 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt
     2341 ctcatcattt tggcaaagaa ttcatgccca agaagaagag gaaggtgtcc aatctcctga
     2401 ctgttcacca gaacctccct gcgctgccag tagatgccac tagcgatgag gtcaggaaaa
     2461 atctcatgga tatgtttagg gatagacagg cgttttctga acacacctgg aaaatgctgc
     2521 ttagcgtgtg ccgatcctgg gcagcctggt gtaagctgaa caatcgcaaa tggttccccg
     2581 ccgagccgga ggacgtgcgc gattacctgc tgtatctcca ggcaagaggg ctggctgtca
     2641 agactatcca gcagcacttg ggccaactga atatgctgca tcgacgcagc gggctccccc
     2701 ggcctagcga ttcaaacgca gtctcccttg ttatgaggag aattagaaag gaaaacgtag
     2761 atgcgggtga gagggctaag caggctctcg cttttgagcg gactgatttc gaccaggtca
     2821 gatccctgat ggataacagt gatcggtgcc aggacatcag gaacctcgca tttctgggaa
     2881 ttgcatataa cacacttctg cgcatagctg agatcgcccg gatcagagtg aaagacatca
     2941 gtcgaacgga cggcggccgg atgcttattc atattggacg cacaaagaca ttggtcagca
     3001 ccgctggcgt tgaaaaggcc ttgtccctgg gcgtaacgaa gctggtggaa agatggatct
     3061 cagtgtccgg cgtggctgac gaccctaata attacttgtt ctgtcgagtg agaaaaaacg
     3121 gagtcgccgc gccctctgcc accagccaat tgagtacacg ggcccttgaa gggatctttg
     3181 aggcaaccca ccgactcata tacggagcca aggatgacag tggccagagg tatctcgcct
     3241 ggtcaggtca ttctgctagg gtgggggccg cacgagacat ggcgcgggca ggagtctcca
     3301 taccagagat tatgcaagct ggaggttgga caaatgtgaa catcgttatg aactatatcc
     3361 gcaatcttga ctctgaaacc ggggccatgg tgagactgct cgaagatggt gacctcgagg
     3421 gaagcggagc cactaacttc tccctgttga aacaagcagg ggatgtcgaa gagaatcccg
     3481 ggccagctag catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3541 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3601 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3661 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3721 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3781 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     3841 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3901 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3961 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4021 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4081 tggtagaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4141 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4201 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4261 atcgccttct tgacgagttc ttctgagcgg ccgcactcct caggtgcagg ctgcctatca
     4321 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc
     4381 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     4441 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     4501 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     4561 aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta
     4621 tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta
     4681 gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt
     4741 acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc
     4801 tcttctctta tggagatccc tcgacctgca gagctccagc ctacacaatc gctcaagacg
     4861 tgtaatgctt ttattatata ttagtcacga tttccatgga tatcaagctt aaacaagaat
     4921 ctctagtttt ctttcttgct tttactttta cttccttaat actcaagtac aattttaatg
     4981 gagtactttt ttacttttac tcaagtaaga ttctagccag atacttttac ttttaattga
     5041 gtaaaatttt ccctaagtac ttgtactttc acttgagtaa aatttttgag tactttttac
     5101 acctctgctc gaccatatgg gagagctccc aacgcgttgg atgcatagct tgagtattct
     5161 atagtgtcac ctaaatagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5221 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5281 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5341 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5401 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5461 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5521 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5581 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5641 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5701 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5761 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5821 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5881 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5941 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6001 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6061 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6121 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6181 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6241 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6301 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6361 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6421 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6481 tgcaatgata ccgcgagaac cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6541 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6601 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6661 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6721 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6781 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6841 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6901 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6961 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7021 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7081 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7141 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7201 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7261 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7321 cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga
     7381 gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     7441 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     7501 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     7561 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     7621 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     7681 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     7741 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     7801 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg
     7861 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     7921 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     7981 cagtcacgac gt
//
