LOCUS       sequence-451637-        6621 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inserts 500bp Prdm8 promoter in place of the Fgf5 promoter along 
            with removable selection cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6621)
  AUTHORS   Jensen CL, Chen LF, Swigut T, Crocker OJ, Yao D, Bassik MC, Ferrell 
            JE Jr, Boettiger AN, Wysocka J
  TITLE     Long-range regulation of transcription scales with genomic distance 
            in a gene-specific manner.
  JOURNAL   Mol Cell. 2025 Jan 16;85(2):347-361.e7. doi: 
            10.1016/j.molcel.2024.10.021. Epub 2024 Dec 2.
  PUBMED    39626660
REFERENCE   2  (bases 1 to 6621)
  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.molcel.2024.10.021"; journalName: "Mol Cell"; date: 
            "2025-01-16- 16"; volume: "85"; issue: "2"; pages: "347-361.e7"
COMMENT     Sequence Label: pCLJ-F2P-LoxP-PGK-PuroR-LoxP
FEATURES             Location/Qualifiers
     source          1..6621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1009..1027
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1009..1026
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    2515..2548
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2550..3149)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2633..2653)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3130..3149
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(3170..3669)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3218..3237)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3611..3634
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    3681..3714
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4731..4740
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4770..4789)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4771..4789)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4796..4813)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4796..4812)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4805..4827)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4953..5408
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5040..5059)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5250..5271
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5486..5590
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5591..5659,5660..6451)
                     /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(5809..5828)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat ttaggtgaca
     1021 ctatagaata ctcaagctat gcatccaacg cgttgggagc tctcccatat ggtcgaccct
     1081 gtgtagttgg tgtgcgcgcg cgcttatgta tgcatgtatg tatctacata tgtatgtatt
     1141 cgtgtgaaag atacctaagc attcggagtt acatagagat attaataaac aaacataata
     1201 tgggatatat aggatatttg tagttggaga atggaagtac tcagatgggt tcttttcata
     1261 aacgtaaatt atgtacaggg aatagagata tacttaacgc ttaaagaaaa atgcggttct
     1321 agttcataag tagaaagaaa ttgtaccagg agaaagattg ttacttcttt cattattatt
     1381 attattattt ttttcttctc cagctagagt gggtggctag tcttttcggc ccgggcattc
     1441 tggaaaagag ctgcctgtct cacctcggta acctcacctg ggcttaccca tgcttactac
     1501 ccaagacaga agggtgcgtc tgctcgctct gagtagacgc atctccacga aaccctaccg
     1561 gactctttcc agaaccagca gagtcccaga cgctagggct cctagaggtg cctgtggccc
     1621 aaaggaatct tgaccgtccc tcccagaaca ggttttgttg gagagtgagc aacttactta
     1681 acacactggc ttcgtgggag ccattgactt tgccatccgg gtagatctgc agatggaaac
     1741 cgatgcccac tctgcagtac aggctgccgg tccggcgccc cgaagggctc cactggaaac
     1801 tgctatgttc cgagccgctt ccttggctgc ccggagaagc tgcgactggt gaggaggcag
     1861 aagacgaaga aaacgtcgcg ctacttctgc cccggctgcc gctggagtct cccgggttcc
     1921 taggaggcgc gggttgccct tcgggagtga gacgcttctc cccgtgagcc caagcgctgt
     1981 ggatcaggtg gctgcagaag atgaggaaga gcaaggacag gctcattctt ccggccgcgg
     2041 gggtcctaag tgcatcttgc agggctggct ccgggctcga gagagagaga gagagttgta
     2101 cagatggacc cgatgcaatg actgactggc acgcagacac acactttttg tttgctgcac
     2161 ataatctgcc caatgacgcg tgacagccca cgtcccctcc ctttaactct tgccggtctg
     2221 ggagatccct ccggcgccac aagcctggtt tttctggact gcccgcgcta ggcagatcct
     2281 aagctccttc ccgaagtggc atagcctccc cgagagcatc ctggcctgcc ttctcctgca
     2341 gctgggtccc tctgggacat ctgggttggc aaaatccgtt tcgggctcag gaaggaaggt
     2401 gtcagaagga gcagagcggt tcttttccgt tttgctgggc actcagagga cgtaggacaa
     2461 ggagttgaat tggctatgtt gccagcgcgt gaactacttg catatgtccc tagaataact
     2521 tcgtataatg tatgctatac gaagttatct taggcaccgg gcttgcgggt catgcaccag
     2581 gtgcgcggtc cttcgggcac ctcgacgtcg gcggtgacgg tgaagccgag ccgctcgtag
     2641 aaggggaggt tgcggggcgc ggaggtctcc aggaaggcgg gcaccccggc gcgctcggcc
     2701 gcctccactc cggggagcac gacggcgctg cccagaccct tgccctggtg gtcgggcgag
     2761 acgccgacgg tggccaggaa ccacgcgggc tccttgggcc ggtgcggcgc caggaggcct
     2821 tccatctgtt gctgcgcggc cagccgggaa ccgctcaact cggccatgcg cgggccgatc
     2881 tcggcgaaca ccgcccccgc ttcgacgctc tccggcgtgg tccagaccgc caccgcggcg
     2941 ccgtcgtccg cgacccacac cttgccgatg tcgagcccga cgcgcgtgag gaagagttct
     3001 tgcagctcgg tgacccgctc gatgtggcgg tccggatcga cggtgtggcg cgtggcgggg
     3061 tagtcggcga acgcggcggc gagggtgcgt acggccctgg ggacgtcgtc gcgggtggcg
     3121 aggcgcaccg tgggcttgta ctcggtcatg gtaagcttgg gctgcaggtc gaaaggcccg
     3181 gagatgagga agaggagaac agcgcggcag acgtgcgctt ttgaagcgtg cagaatgccg
     3241 ggcctccgga ggaccttcgg gcgcccgccc cgcccctgag cccgcccctg agcccgcccc
     3301 cggacccacc ccttcccagc ctctgagccc agaaagcgaa ggagcaaagc tgctattggc
     3361 cgctgcccca aaggcctacc cgcttccatt gctcagcggt gctgtccatc tgcacgagac
     3421 tagtgagacg tgctacttcc atttgtcacg tcctgcacga cgcgagctgc ggggcggggg
     3481 ggaacttcct gactagggga ggagtagaag gtggcgcgaa ggggccacca aagaacggag
     3541 ccggttggcg cctaccggtg gatgtggaat gtgtgcgagg ccagaggcca cttgtgtagc
     3601 gccaagtgcc cagcggggct gctaaagcgc atgctccaga ctgccttggg aaaagcgcct
     3661 cccctaccct cagcggccgc ataacttcgt ataatgtatg ctatacgaag ttatgcacgg
     3721 acggacggca acgtgggcat gggttcccgt cctctgtggt cacatgtgtc ccagccaact
     3781 cactctttac ccagcacccg cattccaggt tgctggcccc tgccatctgg agcctgggaa
     3841 ccactttccg aagggaacca aaaactgatt ggcagcagaa cttgggaagg ttttggggtg
     3901 gcccagggtg ggagagaaga ctggtttctc gctgttattg atttattaaa gaactaaggt
     3961 gtgcgtttat ttctccacta gagagccacc ctgaattgtt atgccaggtc tcaggctgcc
     4021 ctctaagaaa ctctgtggat gcacgacgtg tccagctttt tcttcaggaa aacccatgtc
     4081 tgtgttggaa caggtgaacg ccctcgaaca cacacacaca cacacacaca cacacacaca
     4141 cacacaccag acacacttga actccaggca aggatgaatt gagtctgaac accaggactg
     4201 cccagttgtc ctctctgacc ccatgatccg ttgcctggag gagttagcca acagctctct
     4261 tcaacttcca ttgactggag gaccgaccct aatgaatgat taatgacagg cccctgaatc
     4321 tcaacctagg gaaacctgag aaggaagaat ttccccgtac gtaatgctgg ggctgttatt
     4381 tatctgagaa atatacctga cttgcattct tggaatattt ctgtctgaaa acatatttga
     4441 aactttaaac tacactatga tatatcactc attggtcact taaaatgaaa agtcaatgaa
     4501 gccgcccaat ggggtcaact cgcatcaatc atgcgagatg ttaagacatg actttattaa
     4561 ttactgagta tctgcttcag gcttcctatc ttcatagtga aaacctataa aacagatgta
     4621 gatttgtttt cagaattgca aatcttccgt ggattgggga caaggcagct gatagccagt
     4681 cacctgatca acacaggaaa tcgccaaagg taaacgagac gatcccgcgg ccatggcggc
     4741 cgggagcatg cgacgtcggg cccaattcgc cctatagtga gtcgtattac aattcactgg
     4801 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4861 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     4921 cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc
     4981 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     5041 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5101 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5161 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5221 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5281 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     5341 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     5401 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcatc
     5461 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5521 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5581 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5641 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5701 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5761 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5821 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5881 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5941 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6001 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6061 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6121 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6181 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6241 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6301 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6361 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6421 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6481 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6541 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6601 agaaaagatc aaaggatctt c
//
