LOCUS       sequence-177432-        8163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For lncRNA CAG promoter homologue recombination mediated knockin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8163)
  AUTHORS   Yin Y, Yan P, Lu J, Song G, Zhu Y, Li Z, Zhao Y, Shen B, Huang X, 
            Zhu H, Orkin SH, Shen X
  TITLE     Opposing Roles for the lncRNA Haunt and Its Genomic Locus in 
            Regulating HOXA Gene Activation during Embryonic Stem Cell 
            Differentiation.
  JOURNAL   Cell Stem Cell. 2015 May 7;16(5):504-16. doi: 
            10.1016/j.stem.2015.03.007. Epub 2015 Apr 16.
  PUBMED    25891907
REFERENCE   2  (bases 1 to 8163)
  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"; doi: 
            "10.1016/j.stem.2015.03.007"; journalName: "Cell Stem Cell"; date: 
            "2015-05-7- 7"; volume: "16"; issue: "5"; pages: "504-16"
COMMENT     Sequence Label: PB-CAG-HRKI(Haunt P-Inr2-cDNA KI)
FEATURES             Location/Qualifiers
     source          1..8163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        383..658
                     /label=chicken beta-actin promoter
     intron          659..1667
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1591..1613
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     polyA_signal    2316..2437
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2353..2372)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2407..2426
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2459..2492
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2715..2734)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2716..2734)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2744..2761)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2744..2760)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2753..2775)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2902..3357
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2989..3008)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3199..3220
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3383..3487
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3488..3556,3557..4348)
                     /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(3706..3725)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4519..5107
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5008..5027
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5261..5278
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5395..5416
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5431..5436,5437..5454,5455..5461)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5469..5485
                     /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     5474..5496
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5493..5509
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5493..5509
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5528..5548
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5530..5548
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(6549..7574)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(6549..7574)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(7593..8092)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     8034..8057
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    8115..8148
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg ggtcgaggtg agccccacgt tctgcttcac tctccccatc
      421 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
      481 atgggggcgg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg
      541 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
      601 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
      661 agtcgctgcg ttgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc gcccgccccg
      721 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
      781 ctgtaattag cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct
      841 taaagggctc cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt
      901 gtgtgtgcgt ggggagcgcc gcgtgcggcc cgcgctgccc ggcggctgtg agcgctgcgg
      961 gcgcggcgcg gggctttgtg cgctccgcgt gtgcgcgagg ggagcgcggc cgggggcggt
     1021 gccccgcggt gcgggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg
     1081 gggggtgagc agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct
     1141 ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtgcggg gcgtggcgcg
     1201 gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg
     1261 cctcgggccg gggagggctc gggggagggg cgcggcggcc ccggagcgcc ggcggctgtc
     1321 gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac
     1381 ttcctttgtc ccaaatctgg cggagccgaa atctgggagg cgccgccgca ccccctctag
     1441 cgggcgcggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
     1501 gcgtcgccgc gccgccgtcc ccttctccat ctccagcctc ggggctgccg cagggggacg
     1561 gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct
     1621 ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg
     1681 tgctggttgt tgtgctgtct catcattttg gcaaagaatt cctcgacggt atcgataagc
     1741 tatcacaagt ttgtacattc gaactctcag ctgggagacc gtgggcggag gtgatacaac
     1801 ctgactgatg atggctgaca aaagaagaag aaagtagctg agaaccagca gctgaagaaa
     1861 gcaagaaaat atgagtgaag tcacaaatgt caccaaggaa aggaagaaca gaaaaataac
     1921 ttgaatattc aagatgggca gcctagttca acgacacatc aagccttgtg aaaaggtgac
     1981 tccgagacat ggaggcacat gcctgtagcc tcaccacaca ggcaggtggt ggtgaattct
     2041 acactaacct tggctacaca gtgacacact gccccaaaac agaaacaaaa agcaaaaagt
     2101 ttctcagtga acctcagaaa aggttcctaa ggtaacaaaa gaatcaaaat atgccctagc
     2161 tcaccccaaa tgccaagagc cttgctatgt ttagtaagca cctccataaa atgctgtttt
     2221 cccggaagag agctgaggcc agagaagttg ccaagtgtca atcctaccac atcacgttcc
     2281 tacgtctgcc ctgtcctcca ctcggcacca gcgttaactt gtttattgca gcttataatg
     2341 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2401 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaggt acaagtaggc gcgtcgagat
     2461 aacttcgtat aatgtatgct atacgaagtt atgttttgtt actttataga agaaattttg
     2521 agtttttgtt tttttttaat aaataaataa acataaataa attgtttgtt gaatttatta
     2581 ttagtatgta agtgtaaata taataaaact taatatctat tcaaattaat aaataaacgt
     2641 caattttacg catgattatc tttaacgtac gtcacaatat gattatcttt ctagggttaa
     2701 agagctccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     2761 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     2821 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     2881 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2941 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3001 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3061 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3121 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3181 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3241 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3301 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     3361 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3421 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3481 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     3541 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3601 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     3661 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     3721 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     3781 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     3841 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     3901 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     3961 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4021 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4081 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4141 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4201 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4261 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4321 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4381 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4441 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4501 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4561 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4621 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     4681 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4741 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4801 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4861 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4921 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4981 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5041 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5101 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5161 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5221 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5281 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     5341 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     5401 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     5461 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     5521 caagcgcgca attaaccctc actaaaggga acaaaagctg ggtacctgag aacttcagtg
     5581 ggatacattg agctgcttta agcatcaggg agtagaacct gtgtgcactg ccatagtgct
     5641 tcatttattt agacctaata gaatcagatc agtgtctctc catttacctt tgccatgctt
     5701 tcaagacatt agaaacaagc tcttttgact tcagagacac aacaattaga tataaaacac
     5761 tagccttgag tggatcttgg actgtttgaa agcgtcctga caggtaagtg atttttctat
     5821 atgcttgaaa gttttcaaaa taaaaacttt gaaggaaggg ctcttttcct tgtccttcac
     5881 ttagccgtgc tacagggtgt ccggacagga taatgtatga ggactgatgt acccttttga
     5941 cctggtggtg tgctttttca ggcaggttgc ttactgccca agggctccat aactggggag
     6001 gacacaatga gaaatcaaac cctcacaaac atatcgagga gggttatcaa cacctcagcc
     6061 aacatgcatt cttgaaatat tgctctctct ttctaaatag cgcgaatccg tcgctgtgca
     6121 tttaggacat ctcagtcgcc gcttggagct cccgtgaggc gtgcttgtca atgcggtaag
     6181 tgtcactgat tttgaactat aacgaccgcg tgagtcaaaa tgacgcatga ttatctttta
     6241 cgtgactttt aagatttaac tcatacgata attatattgt tatttcatgt tctacttacg
     6301 tgataactta ttatatatat attttcttgt tatagatatc caactatgaa acattatcat
     6361 aaagcaatag taaagagcct tcagtaaaga gcaggcattt atctaatccc accccacccc
     6421 cacccccgta gctccaatcc ttccattcaa aatgtaggta ctctgttctc acccttctta
     6481 acaaagtatg acaggaaaaa acttccatgt ggacatcttt attgtttaat agatcatcaa
     6541 tttctcgact attcctttgc cctcggacga gtgctggggc gtcggtttcc actatcggcg
     6601 agtacttcta cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc
     6661 ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca
     6721 tcatcgaaat tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata
     6781 tacgcccgga gccgcggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc
     6841 tgctgctcca tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg
     6901 gaatccccga acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc
     6961 aggacattgt tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg
     7021 gcccaaagca tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc
     7081 acagtttgcc agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta
     7141 gtgtattgac cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg
     7201 gccgcagcga tcgcatccat ggcctccgcg accggctgca gaacagcggg cagttcggtt
     7261 tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc
     7321 tcgctgaatt ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc
     7381 cgataaacat aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat
     7441 ccacgccctc ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc
     7501 aggtcggaga cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt
     7561 tcaggctttt tcatggtggc ggctggatcg gtcgaaaggc ccggagatga ggaagaggag
     7621 aacagcgcgg cagacgtgcg cttttgaagc gtgcgagaat gccgggcctc cggaggacct
     7681 tcgggcgccc gccccgcccc tgagcccgcc cctgagcccg cccccggacc caccccttcc
     7741 cagcctctga gcccagaaag cgaaggagca aagctgctat tggccgctgc cccaaaggcc
     7801 tacccgcttc cattgctcag cggtgctgtc catctgcacg agactagtga gacgtgctac
     7861 ttccatttgt cacgtcctgc acgacgcgag ctgcggggcg ggggggaact tcctgactag
     7921 gggaggagta gaaggtggcg cgaaggggcc accaaagaac ggagccggtt ggcgcctacc
     7981 ggtggatgtg gaatgtgtgc gaggccagag gccacttgtg tagcgccaag tgccagcggg
     8041 gctgctaaag cgcatgctcc agactgcctt gggaaaagcg cctcccctac ccggtagaat
     8101 tcatcgatga tatcataact tcgtataatg tatgctatac gaagttatgc ggccctcgag
     8161 gtc
//
