LOCUS       sequence-366140-        7180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA with AU flip (sgRNA2.0), mKO2 as selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7180)
  AUTHORS   Chan WF, Coughlan HD, Iannarella N, Smyth GK, Johanson TM, Keenan 
            CR, Allan RS
  TITLE     Identification and characterization of the long noncoding RNA Dreg1 
            as a novel regulator of Gata3.
  JOURNAL   Immunol Cell Biol. 2020 Sep 24. doi: 10.1111/imcb.12408.
  PUBMED    32970351
REFERENCE   2  (bases 1 to 7180)
  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: "Immunol 
            Cell Biol. 2020 Sep 24. doi: 10.1111/imcb.12408."
COMMENT     Sequence Label: Sp(AU)sgRNA-mKO2
FEATURES             Location/Qualifiers
     source          1..7180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     420..438
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      518..527
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             524..1180
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     LTR             1286..1519
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1591..1712
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1628..1647)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1682..1701
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1752..1887
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1907..1926)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1908..1926)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1936..1953)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1936..1952)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1945..1967)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2094..2549
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2181..2200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2391..2412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2575..2679
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2680..2748,2749..3540)
                     /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     complement(2898..2917)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3711..4299
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4200..4219
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4453..4470
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4587..4608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4623..4628,4629..4646,4647..4653)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4661..4677
                     /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     4666..4688
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4685..4701
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4685..4701
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4720..4740
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4722..4740
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4768..4994
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4995..5175
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5222..5347
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5840..6073
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6258..6302
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        6600..6848
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6600..6620
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6771..6790
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    7005..7122
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7171..7180,1..501)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt
       61 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc
      121 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc
      181 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca
      241 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg
      301 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa
      361 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg
      421 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga
      481 atcaccgacc tctctcccca gggggatcct taatgccgcc accatggtga gtgtgattaa
      541 accagagatg aagatgaggt actacatgga cggctccgtc aatgggcatg agttcacaat
      601 tgaaggtgaa ggcacaggca gaccttacga gggacatcaa gagatgacac tacgcgtcac
      661 aatggccgag ggcgggccaa tgcctttcgc gtttgactta gtgtcacacg tgttctgtta
      721 cggccacaga gtatttacta aatatccaga agagatacca gactatttca aacaagcatt
      781 tcctgaaggc ctgtcatggg aaaggtcgtt ggagttcgaa gatggtgggt ccgcttcagt
      841 cagtgcgcat ataagcctta gaggaaacac cttctaccac aaatccaaat ttactggggt
      901 taactttcct gccgatggtc ctatcatgca aaaccaaagt gttgattggg agccatcaac
      961 cgagaaaatt actgccagcg acggagttct gaagggtgat gttacgatgt acctaaaact
     1021 tgaaggaggc ggcaatcaca aatgccaaat gaaaactact tacaaggcgg caaaagagat
     1081 tcttgaaatg ccaggagacc attacatcgg ccatcgcctc gtcaggaaaa ccgaaggcaa
     1141 cattactgag caggtagaag atgcagtagc tcattcctaa agtagatgcc gacgtcgacc
     1201 tcgagggggg gcccggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     1261 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagaaaagat
     1321 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     1381 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     1441 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     1501 gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt
     1561 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
     1621 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     1681 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
     1741 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     1801 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     1861 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc
     1921 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     1981 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     2041 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     2101 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2161 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2221 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2281 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2341 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2401 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2461 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2521 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     2581 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2641 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2701 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2761 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2821 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2881 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     2941 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3001 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3061 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3121 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3181 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3241 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3301 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3361 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3421 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3481 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3541 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3601 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3661 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3721 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3781 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3841 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     3901 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3961 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4021 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4081 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4141 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4201 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4261 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4321 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4381 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4441 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     4501 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4561 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     4621 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     4681 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
     4741 gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca
     4801 acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc
     4861 gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga
     4921 catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag
     4981 ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct
     5041 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     5101 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     5161 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
     5221 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
     5281 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
     5341 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
     5401 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
     5461 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
     5521 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
     5581 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
     5641 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca
     5701 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     5761 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     5821 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     5881 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     5941 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     6001 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     6061 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     6121 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     6181 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     6241 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     6301 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     6361 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     6421 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca
     6481 ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag
     6541 tgaacggatc tcgacggtat cgatcacgag actagcctcg agcggccgcc cccttcaccg
     6601 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     6661 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     6721 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     6781 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     6841 gaaacaccgg tgtcttcgag acgaagaccc gtttaagagc tatgctggaa acagcatagc
     6901 aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     6961 tttgaattct cgacctcgag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     7021 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     7081 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     7141 agcagagatc cactttggcc gcggctcgag ggggttgggg
//
