LOCUS       sequence-9915-ex        8318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8318)
  AUTHORS   Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R
  TITLE     Efficient method to generate single-copy transgenic mice by 
            site-specific integration in embryonic stem cells.
  JOURNAL   Genesis. 2006 Jan . 44(1):23-8.
  PUBMED    16400644
REFERENCE   2  (bases 1 to 8318)
  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: "Genesis. 
            2006 Jan . 44(1):23-8."
COMMENT     Sequence Label: CAGGS-losSTOPlox-ClaI flp-in
FEATURES             Location/Qualifiers
     source          1..8318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        3..382
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..661
                     /label=chicken beta-actin promoter
     intron          663..1680
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1604..1626
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1688..1707
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1740..1756
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1770..1803
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1833..2329
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             2343..3146
                     /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(2406..2425)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3016..3035
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3156..3617
                     /label=PGK poly(A) signal
                     /note="mouse phosphoglycerate kinase 1 polyadenylation 
                     signal"
     polyA_signal    3765..3899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4019..4153
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4273..4407
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    4436..4469
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    4529..4650
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4748..4764)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4774..4821)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        complement(4863..5362)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5402..5418)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5450..5469)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5451..5469)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5479..5496)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5479..5495)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5488..5510)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5637..6092
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5724..5743)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5934..5955
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6118..6222
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6223..6291,6292..7083)
                     /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(6441..6460)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7254..7842
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7743..7762
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7996..8013
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8130..8151
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8166..8171,8172..8189,8190..8196)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8204..8220
                     /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     8209..8231
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8228..8244
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8228..8244
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8263..8283
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8265..8283
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
       61 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      121 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      181 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      241 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      301 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      361 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
      421 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
      481 gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 ggggagtcgc tgcgacgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      721 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      781 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      841 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
      961 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     1021 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     1081 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     1141 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     1201 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     1261 gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc
     1321 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     1381 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     1441 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     1501 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     1561 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1621 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1681 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attggccgct
     1741 ctagaactag tggatccgga acccttaata taacttcgta taatgtatgc tatacgaagt
     1801 tattaggtcc ctcgacctgc aggaattcta ccgggtaggg gaggcgcttt tcccaaggca
     1861 gtctggagca tgcgctttag cagccccgct ggcacttggc gctacacaag tggcctctgg
     1921 cctcgcacac attccacatc caccggtagc gccaaccggc tccgttcttt ggtggcccct
     1981 tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt
     2041 cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca
     2101 ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct
     2161 tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc
     2221 tcaggggcgg ggcgggcgcg aaggtcctcc cgaggcccgg cattctcgca cgcttcaaaa
     2281 gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcga cctgcagcca
     2341 atatgggatc ggccattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     2401 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     2461 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     2521 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     2581 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     2641 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     2701 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     2761 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     2821 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     2881 gcatgcccga cggcgatgat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     2941 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     3001 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     3061 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     3121 atcgccttct tgacgagttc ttctgagggg atccgctgta agtctgcaga aattgatgat
     3181 ctattaaaca ataaagatgt ccactaaaat ggaagttttt cctgtcatac tttgttaaga
     3241 agggtgagaa cagagtacct acattttgaa tggaaggatt ggagctacgg gggtgggggt
     3301 ggggtgggat tagataaatg cctgctcttt actgaaggct ctttactatt gctttatgat
     3361 aatgtttcat agttggatat cataatttaa acaagcaaaa ccaaattaag ggccagctca
     3421 ttcctcccac tcatgatcta tagatctata gatctctcgt gggatcattg tttttctctt
     3481 gattcccact ttgtggttct aagtactgtg gtttccaaat gtgtcagttt catagcctga
     3541 agaacgagat cagcagcctc tgttccacat acacttcatt ctcagtattg ttttgccaag
     3601 ttctaattcc atcagaagct tgcagatctg cgactctaga ggatctgcga ctctagagga
     3661 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     3721 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     3781 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3841 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatct
     3901 gcgactctag aggatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     3961 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     4021 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4081 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4141 tcatgtctgg atctgcgact ctagaggatc ataatcagcc ataccacatt tgtagaggtt
     4201 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     4261 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4321 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4381 atcaatgtat cttatcatgt ctggatcccc atcaagctga tccggaaccc ttaatataac
     4441 ttcgtataat gtatgctata cgaagttatt aggtccctcg acctgcagcc caagctagct
     4501 tatcgatggc cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     4561 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     4621 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     4681 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     4741 aaaatcggat ccactagttc tagagcggcc atcgaagttc ctatactttc tagagaatag
     4801 gaacttcgga atagtaactt ctccataagc ttcagctgct cgagatctag atggatgcag
     4861 gtcgaaaggc ccggagatga ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc
     4921 gtgcagaatg ccgggcctcc ggaggacctt cgggcgcccg ccccgcccct gagcccgccc
     4981 ctgagcccgc ccccggaccc accccttccc agcctctgag cccagaaagc gaaggagcaa
     5041 agctgctatt ggccgctgcc ccaaaggcct acccgcttcc attgctcagc ggtgctgtcc
     5101 atctgcacga gactagtgag acgtgctact tccatttgtc acgtcctgca cgacgcgagc
     5161 tgcggggcgg gggggaactt cctgactagg ggaggagtag aaggtggcgc gaaggggcca
     5221 ccaaagaacg gagccggttg gcgcctaccg gtggatgtgg aatgtgtgcg aggccagagg
     5281 ccacttgtgt agcgccaagt gcccagcggg gctgctaaag cgcatgctcc agactgcctt
     5341 gggaaaagcg cctcccctac ccggtagaat ttcgaggtcg acgaattcct gcagcccggg
     5401 ggatccacta gttctagagc ggccgccacc gcggtggagc tccaattcgc cctatagtga
     5461 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     5521 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     5581 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     5641 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5701 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5761 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5821 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     5881 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5941 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     6001 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     6061 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     6121 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     6181 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6241 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6301 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6361 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6421 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     6481 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     6541 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6601 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6661 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6721 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6781 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6841 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6901 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6961 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     7021 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     7081 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     7141 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     7201 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7261 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7321 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7381 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     7441 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     7501 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     7561 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7621 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7681 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7741 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7801 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7861 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     7921 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     7981 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     8041 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     8101 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     8161 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     8221 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     8281 agggaacaaa agctgggtac cgggcccccc ctcgagat
//
