LOCUS       sequence-265185-        5749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  STITCH (Synthetic Toplogical Insulator with TetO for 
            CHromatin-engineering) with homology arms for MYC locus insertion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5749)
  AUTHORS   Tsujimura T, Takase O, Yoshikawa M, Sano E, Hayashi M, Hoshi K, 
            Takato T, Toyoda A, Okano H, Hishikawa K
  TITLE     Controlling gene activation by enhancers through a drug-inducible 
            topological insulator.
  JOURNAL   Elife. 2020 May 5;9. pii: 47980. doi: 10.7554/eLife.47980.
  PUBMED    32369019
REFERENCE   2  (bases 1 to 5749)
  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: "Elife."; 
            date: "2020-05-5"; volume: "9. pii: 47980. doi"; pages: " 
            10.7554/eLife.47980"
COMMENT     Sequence Label: pUC-STITCH
FEATURES             Location/Qualifiers
     source          1..5749
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    641..659
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1379..1397
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1400..1433
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1448..1947
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1483..1506)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1880..1899
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(2621..2640)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2675..2694
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2707..2740
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2743..2761
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3303..3321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(3528..3544)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3528..3544)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3541..3563)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3552..3568
                     /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(3576..3582,3583..3600,3601..3606))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3621..3642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3759..3776)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3930..4518)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4010..4029)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4689..5480,5481..5549))
                     /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     5312..5331
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5550..5654)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5722..5740
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accaaatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcatcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgcaac gcgatgacga tggatagcga
      421 ttcatcgatg agctgacccg atcgccgccg ccggagggtt gcgtttgaga cgggcgacag
      481 atggatccaa aataaagttg tatgggaaaa gcattataca caagctggaa aaatgacaat
      541 aagacacata aaggaaaagg gactgtggaa ctttccctgg ggaagagtat atgtccagtc
      601 ccaccccaga tatcagcctc cagccccctg gttgtcaaac tccctatcag tgatagagaa
      661 cgacccagca aagccatccc attccaagat gtgaatggac cttggagcca ctccccaccc
      721 ccaccactcc caagcacagc cattcagtgc ctctccctgg gcaggggaac gctgccctct
      781 tctggtccaa gcggacaggc acggccaagg ccagcctcca gcacttcatg agccgcctgc
      841 cactggtttg tatagtctga gggtcatgcc aaccctctgc ctctcaagtc cttttaagga
      901 aaacgacagg gcacctttct gtgcgccacc tggtggcaag aattgtcatt acaccagcca
      961 ggagattctc ctgccccaga acagaccatt gacctgtcct gagggagggt ggctcttccg
     1021 acttgcttgc tgttctcgtt gataacaaga acaattgggc tcaattccaa acttctatta
     1081 ggcacctccc aagcaccatt aagctccagt ttcagggcct ctggtgccct cttctggcct
     1141 gcacatgcac tgcacatgta tggcacacat acaaacaagt atacacacac tttttttacg
     1201 caaggcggtt aaagcctagc caggtagacg ccacagaatt ccgtgcaagt tccagcagct
     1261 ggaggccccg ctgggatgga tgcgcgctac tgcggccccc tctgctggca gcctcaaggc
     1321 tgcgccttcc ggggaggtgg ccaagcagga ggctgcacag ccaagccctc agaccgactc
     1381 cctatcagtg atagagaaca taacttcgta taatgtatgc tatacgaagt tatctcgaaa
     1441 ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc
     1501 ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc
     1561 ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc
     1621 ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa
     1681 gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta
     1741 ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg
     1801 ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga
     1861 aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc
     1921 tcctcttcct catctccggg cctttcgacc tgcatccatc tagatctcga gcagctgaag
     1981 cttaccatga ccgagtacaa gccaaccgtg cgactggcca cgagggatga cgtccccaga
     2041 gccgttcgta cgctggctgc tgcctttgcg gactatcctg ccacaagaca caccgtcgat
     2101 ccggaccggc atatcgaacg cgtgaccgaa ctgcaggagc tgttcctgac acgagtcggc
     2161 ttggacatag ggaaggtatg ggtggcagat gatggtgccg ctgttgccgt ctggacaacc
     2221 ccggaaagcg tggaagcagg agcagtgttc gctgagattg gacccagaat ggccgagctc
     2281 tctgggagta ggttggccgc tcaacagcag atggaaggcc tgcttgcccc acatcggcct
     2341 aaggagcctg cgtggtttct ggcgactgtt ggcgtgtcac ccgatcacca gggtaaaggg
     2401 cttggcagcg ctgtggtgct cccaggagtc gaagcagcag agagagctgg tgtacccgcc
     2461 tttctggaga cttccgcacc tcgcaacctc cccttctacg agaggctggg cttcaccgtt
     2521 actgccgacg tggaggtgcc tgaaggacca cggacatggt gcatgactcg caaaccaggg
     2581 gcttgattgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     2641 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2701 tcttatataa cttcgtataa tgtatgctat acgaagttat actccctatc agtgatagag
     2761 aacttcccat taagagggac cacacccgag cggctgagtg ttttggcttg aatgtggcag
     2821 gagcaccctc cccactcagg ctcctcctgt gttagaacac ttggacccca gcagggggca
     2881 gtgttggaag gttgtagaac cttcacagcg gaggccgtgg gtagccttca ttagtgccaa
     2941 gaaggccacc ccagtgtggg gagcacaagg cctgccatgc tgaggaagga aaacagccta
     3001 gcagccaaga gtgctgctgc agcatccgtt catccagcag agggcagcag agagcagaag
     3061 ttgcctcagg gaggaacctt gggctgttag cactaggggt tgttggacac tgtcccctct
     3121 cagggacaga atgtttgacg tgcagagggg ctactcagcc ctctgcagct cctgttggct
     3181 gtcatcattc tgggcctgcg gggcaggaaa actcaaccac cagcaggggg cgcctgtgac
     3241 ctttgacttc actgtgcagt cctgagccca acaggacaca tgggtttgct tgctctcttg
     3301 actccctatc agtgatagag aacactctgg gactggtgca gtgacctcac cagaaggcat
     3361 gagatccagc gaattattca gaatattggt tctgcttcgg atgagatgcc agtgagaata
     3421 tcaccagcta actgcaggca gggaggaagt caatggtaat taaactttgt ttcggatcca
     3481 tcagttctgg accagcgagc tgtgctgcga ctcgtggcgt aatcatggtc atagctgttt
     3541 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3601 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3661 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3721 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3781 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3841 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3901 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3961 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4021 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgtc gcttaccgga
     4081 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4141 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4201 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4261 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4321 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4381 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4441 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4501 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4561 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4621 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4681 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4741 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4801 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4861 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4921 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4981 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5041 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5101 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5161 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5221 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5281 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5341 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5401 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5461 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5521 agggcgacac ggaaatgttg aatactcata ctctaccttt ttcaatatta ttgaagcatt
     5581 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5641 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     5701 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
