LOCUS       sequence-316186-        4793 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  F3 (FRT variant)-flanked pPgk-pEM7-Neo*-bpA positive selection 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4793)
  AUTHORS   Nam HS, Capecchi MR
  TITLE     Additional gene targeting vector construction plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4793)
  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: 
            "Unpublished"
COMMENT     Sequence Label: F3 Neo* F3
FEATURES             Location/Qualifiers
     source          1..4793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..500
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(36..59)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     433..452
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        512..559
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             578..1381
                     /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
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(641..660)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1251..1270
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1413..1430)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1419..1643
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    1698..1731
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     primer_bind     complement(1751..1767)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1751..1767)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(1804..1822)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1843..1859)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1843..1859)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1856..1878)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1867..1883
                     /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(1891..1897,1898..1915,1916..1921))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1936..1957
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2074..2091)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2245..2833)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2325..2344)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3004..3795,3796..3864))
                     /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     3627..3646
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3865..3969)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3995..4450)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4132..4153)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4344..4363
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4577..4599
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4591..4608
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4592..4608
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4615..4634
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4615..4633
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4658..4674
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4659..4675
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4697..4730
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
ORIGIN
        1 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
       61 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
      121 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
      181 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
      241 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
      301 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
      361 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
      421 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
      481 cctcatctcc gggcctttcg acctgcagcc tgttgacaat taatcatcgg catagtatat
      541 cggcatagta taatacgaca aggtgaggaa ctaaaccatg ggatcggcca ttgaacaaga
      601 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
      661 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
      721 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
      781 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
      841 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
      901 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
      961 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     1021 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     1081 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg atgatctcgt
     1141 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     1201 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     1261 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     1321 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     1381 aggggatcaa ttctctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     1441 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     1501 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     1561 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     1621 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgactag
     1681 agcttgcgga acccttcgaa gttcctattc ttcaaatagt ataggaactt catcagtcag
     1741 gtacataatg gatccactag ttctagagcg gccgccaccg cggtggagct ccagcttttg
     1801 ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt
     1861 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     1921 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     1981 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2041 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2101 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     2161 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2221 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2281 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2341 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2401 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2461 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2521 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     2581 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     2641 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     2701 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     2761 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     2821 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     2881 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     2941 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3001 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3061 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3121 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3181 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3241 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3301 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3361 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3421 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3481 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3541 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3601 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3661 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3721 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3781 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3841 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     3901 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3961 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     4021 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     4081 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     4141 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     4201 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4261 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4321 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4381 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4441 cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     4501 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     4561 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac
     4621 gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgacgg tatcgataag
     4681 cttgatatcg aattccgaag ttcctattct tcaaatagta taggaacttc aggtctgaag
     4741 aggagtttac gtccagccaa gctagcttgg ctgcaggtcg tcgaaattct acc
//
