LOCUS       sequence-451641-        6910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inserts 500bp Fgf5 promoter in place of the Prdm8 promoter along 
            with removable selection cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6910)
  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 6910)
  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-P2F-Frt-PGK-Puro-Frt
FEATURES             Location/Qualifiers
     source          1..6910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1063..1096
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        1103..1602
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1138..1161)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1535..1554
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1615..2217
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MATEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     2114..2134
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2245..2262)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2251..2458
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2467..2500
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        complement(4035..4053)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4036..4053)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4071..4087)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4071..4087)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4084..4106)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4095..4111
                     /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(4119..4125,4126..4143,4144..4149))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4164..4185
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4302..4319)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4473..5061)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4553..4572)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5232..6023,6024..6092))
                     /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     5855..5874
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6093..6197)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6275..6730)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6412..6433)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6624..6643
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6856..6878
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6870..6887
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6871..6887
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6894..6910,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6894..6910,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggccgcggga tcgtctcgtt
       61 taccagacag gagtctcaga gctccgtaaa gattatactg tcagctgctc tttgccaacc
      121 ctcatatgct cactctgaga ggcaaggaca gagattgtct cctccttaaa ttctggccct
      181 gactcctgcc ctttagcgag taaactatac aattggattt atactcaacc tgacctattg
      241 ctcaattgaa ttccgggaga gtgggggtag gaattgaggg attgcctgcg ccactcaaag
      301 gcctttcttt ccttaggaat agaaagccta ccagagaaaa acagccagag agcccctagc
      361 gtcattgttt tgtggccccc gcagaaggag aaaactctct ggctgtcaaa gacctcaagt
      421 cattagcatc agctgccgct ccagaggaca agccagcgcc tctaagtggc ttagcgcaca
      481 aacgagactc ccgagacagc ctcactcctg tgattcggtc cactcctcca tcaacggctg
      541 gatgtgaaag gctcttgaga gtccttctca gtaatttttt ttttaacttt gaaccatcct
      601 ttctgtatca tgaggtctta gaatcctggc cgccattatg ggaccccctt gggaaccaca
      661 aaatacagag agagcacaga aggtgaaaag ctttaacacc tgagtccaat gacctaggct
      721 tttcccagca acgccctcct agcccatatc cacgcacaga cccttgttct gggacctaaa
      781 aacagccgaa gtccctttag gtgtctggca gcattccaga cttatcttct taccccatcc
      841 cattggatgg gagatggcta gccgagttcc caccaaatca taccccctgt cgccagtatt
      901 tacacgggag tctctgctag gtggcatgaa tgcattttaa ggaagtctgt ttgtcacctc
      961 ctaggccact agcttcacag atcagcggca gtcaatggcc aaagaatccc gcggggcaaa
     1021 ttctcaccct ccttttgggg gagcagggac ataccactca aagaagttcc tattctctag
     1081 aaagtatagg aacttctcta ccgggtaggg gaggcgcttt tcccaaggca gtctggagca
     1141 tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg gcctcgcaca
     1201 cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc cttcgcgcca
     1261 ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg
     1321 acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag caccgctgag
     1381 caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc cttcgctttc
     1441 tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc
     1501 ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg
     1561 tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcag cccaatggcc
     1621 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc
     1681 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     1741 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     1801 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     1861 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     1921 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     1981 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     2041 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     2101 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     2161 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctaagaa
     2221 ttctaactag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2281 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2341 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2401 gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc
     2461 ggccgcgaag ttcctattct ctagaaagta taggaacttc cccttgcgtg gagagcagcg
     2521 cgcgcggagc cgattccgga gccgctggct cgggcagggg gtggggtgac tgggtggctc
     2581 ttgacaaccg ctgggttgca tgacccgtgc acagtcgcag ccagcgtccc gggggcagct
     2641 tctgagggcg agacacctcc tcccactccc gggatcgcat ctccctcctc cttcacaccc
     2701 ccgcccccgg gaccgcggcg ctccctcccc gtaccggccg tgagtacaca aagcggcggg
     2761 tgaggggaag cttcgcaggc gtgcacggag cagtgagatc actggcgtta taaatatccc
     2821 ggtgccagcg ccgagatccg ctcgggtggc ctctctctct ccccctctcc ctctcccttc
     2881 cccgaggcta tgtccaccct gtgcggcgag ggaggcagcg ccagaggcac gcagccgcgc
     2941 gggggctacg ctctctctct ctcctaatct atctgcatct ctcttctctc tttggacaga
     3001 acccacaaga ctgacaccga cacctcttga catggagata cactaatgta ataggcagga
     3061 aggaacactg gcttgcaccc cgttggttcc aggtggcctt atttgggcga ctcggtcctg
     3121 accttatttc tggtaagtct atttgctttg acctgggagg gggatgggaa gaggacaggt
     3181 tgagtacaag gagtgaaaaa attgtccccc ttttcctcgt tatctagaag ataggaggga
     3241 aacgacgcgg aatgttgttt ccactgccaa taagattata caaagggagg agcgggtagg
     3301 agcacttttc tggagaagct agggactggg gagctcgcgg ctgccagaca atgccagccc
     3361 ggagtggagg gagagcatcc ggcagtagcg accggctttc agcgagccca gctggctctg
     3421 agtttcccag gaggatctgc gaaggaagag aacccgggcc tagggatggg cgtaggactg
     3481 tgaaaggcta gatactataa agcccggggt cctgcctatc gctattgtgg ctgatttgcg
     3541 tcaaggcggc caccgaggca gcatggaccg ctattagccc ggctgtggga ccttttcgct
     3601 gtattgttag atagaactgc attttaatga ctgagtccct gctgttgcca aagtggaggg
     3661 cgatctcctg tcacaataaa aagcttctgt gaagaatact ttcaaacgcg aggcagaaat
     3721 caaggtacaa accgttgaaa tgtaggcaac tgggaaggga aagagggttt atactgaaag
     3781 ggagctgaga gaacctgtat tctgcaacgt atttgacttg caattggaaa tcagaagggg
     3841 acaggacgag tgcagaaaca gaaagtagta tcataaactt tctatgttgt ggctattttt
     3901 cttattccca ctgtctggaa aatatacttg ccatttgggg catatgggag accgggagac
     3961 gatcactagt gcggccgcct gcaggtcgac catatgggag agctcccaac gcgttggatg
     4021 catagcttga gtattctata gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg
     4081 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4141 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4201 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4261 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4321 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4381 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4441 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4501 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4561 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4621 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4681 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4741 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4801 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4861 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4921 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4981 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5041 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5101 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5161 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5221 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5281 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5341 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5401 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5461 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5521 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5581 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5641 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5701 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5761 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5821 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5881 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5941 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6001 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6061 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6121 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6181 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgatgcggt gtgaaatacc
     6241 gcacagatgc gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta
     6301 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     6361 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     6421 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     6481 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     6541 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     6601 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     6661 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     6721 cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     6781 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     6841 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg
     6901 actcactata
//
