LOCUS       sequence-451645-        6820 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 6820)
  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 6820)
  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-NeoR-LoxP
FEATURES             Location/Qualifiers
     source          1..6820
                     /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    2513..2546
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2548..3351)
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2659..2678)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3269..3288
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3369..3868)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3417..3436)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3810..3833
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    3880..3913
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4930..4939
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4969..4988)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4970..4988)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4995..5012)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4995..5011)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5004..5026)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5152..5607
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5239..5258)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5449..5470
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5685..5789
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5790..5858,5859..6650)
                     /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(6008..6027)
                     /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 gagttgtaca
     2101 gatggacccg atgcaatgac tgactggcac gcagacacac actttttgtt tgctgcacat
     2161 aatctgccca atgacgcgtg acagcccacg tcccctccct ttaactcttg ccggtctggg
     2221 agatccctcc ggcgccacaa gcctggtttt tctggactgc ccgcgctagg cagatcctaa
     2281 gctccttccc gaagtggcat agcctccccg agagcatcct ggcctgcctt ctcctgcagc
     2341 tgggtccctc tgggacatct gggttggcaa aatccgtttc gggctcagga aggaaggtgt
     2401 cagaaggagc agagcggttc ttttccgttt tgctgggcac tcagaggacg taggacaagg
     2461 agttgaattg gctatgttgc cagcgcgtga actacttgca tatgtcccta gaataacttc
     2521 gtataatgta tgctatacga agttatctca gaagaactcg tcaagaaggc gatagaaggc
     2581 gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
     2641 gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
     2701 cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
     2761 cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt
     2821 gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
     2881 atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
     2941 gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
     3001 ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
     3061 caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
     3121 gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc
     3181 ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
     3241 ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
     3301 agcggccgga gaacctgcgt gcaatccatc ttgttcaatg gccgatccca taagcttggg
     3361 ctgcaggtcg aaaggcccgg agatgaggaa gaggagaaca gcgcggcaga cgtgcgcttt
     3421 tgaagcgtgc agaatgccgg gcctccggag gaccttcggg cgcccgcccc gcccctgagc
     3481 ccgcccctga gcccgccccc ggacccaccc cttcccagcc tctgagccca gaaagcgaag
     3541 gagcaaagct gctattggcc gctgccccaa aggcctaccc gcttccattg ctcagcggtg
     3601 ctgtccatct gcacgagact agtgagacgt gctacttcca tttgtcacgt cctgcacgac
     3661 gcgagctgcg gggcgggggg gaacttcctg actaggggag gagtagaagg tggcgcgaag
     3721 gggccaccaa agaacggagc cggttggcgc ctaccggtgg atgtggaatg tgtgcgaggc
     3781 cagaggccac ttgtgtagcg ccaagtgccc agcggggctg ctaaagcgca tgctccagac
     3841 tgccttggga aaagcgcctc ccctaccctc agcggccgca taacttcgta taatgtatgc
     3901 tatacgaagt tatgcacgga cggacggcaa cgtgggcatg ggttcccgtc ctctgtggtc
     3961 acatgtgtcc cagccaactc actctttacc cagcacccgc attccaggtt gctggcccct
     4021 gccatctgga gcctgggaac cactttccga agggaaccaa aaactgattg gcagcagaac
     4081 ttgggaaggt tttggggtgg cccagggtgg gagagaagac tggtttctcg ctgttattga
     4141 tttattaaag aactaaggtg tgcgtttatt tctccactag agagccaccc tgaattgtta
     4201 tgccaggtct caggctgccc tctaagaaac tctgtggatg cacgacgtgt ccagcttttt
     4261 cttcaggaaa acccatgtct gtgttggaac aggtgaacgc cctcgaacac acacacacac
     4321 acacacacac acacacacac acacaccaga cacacttgaa ctccaggcaa ggatgaattg
     4381 agtctgaaca ccaggactgc ccagttgtcc tctctgaccc catgatccgt tgcctggagg
     4441 agttagccaa cagctctctt caacttccat tgactggagg accgacccta atgaatgatt
     4501 aatgacaggc ccctgaatct caacctaggg aaacctgaga aggaagaatt tccccgtacg
     4561 taatgctggg gctgttattt atctgagaaa tatacctgac ttgcattctt ggaatatttc
     4621 tgtctgaaaa catatttgaa actttaaact acactatgat atatcactca ttggtcactt
     4681 aaaatgaaaa gtcaatgaag ccgcccaatg gggtcaactc gcatcaatca tgcgagatgt
     4741 taagacatga ctttattaat tactgagtat ctgcttcagg cttcctatct tcatagtgaa
     4801 aacctataaa acagatgtag atttgttttc agaattgcaa atcttccgtg gattggggac
     4861 aaggcagctg atagccagtc acctgatcaa cacaggaaat cgccaaaggt aaacgagacg
     4921 atcccgcggc catggcggcc gggagcatgc gacgtcgggc ccaattcgcc ctatagtgag
     4981 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     5041 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     5101 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gacgcgccct
     5161 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5221 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5281 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5341 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     5401 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5461 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     5521 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5581 ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg
     5641 cggtatttca caccgcatca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5701 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5761 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5821 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5881 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5941 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6001 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6061 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6121 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6181 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6241 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6301 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6361 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6421 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6481 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6541 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6601 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6661 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6721 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6781 actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
