LOCUS       sequence-11443-e        5274 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 5274)
  AUTHORS   Singla V, Hunkapiller J, Santos N, Seol AD, Norman AR, Wakenight P, 
            Skarnes WC, Reiter JF
  TITLE     Floxin, a resource for genetically engineering mouse ESCs.
  JOURNAL   Nat Methods. 2010 Jan . 7(1):50-2.
  PUBMED    19966808
REFERENCE   2  (bases 1 to 5274)
  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: "Nat 
            Methods. 2010 Jan . 7(1):50-2."
COMMENT     Sequence Label: pFloxin-IRES-eGFP
FEATURES             Location/Qualifiers
     source          1..5274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      520..529
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(532..565)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    879..1451
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    902..1453
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1046..1063)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     regulatory      1500..1509
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1506..1508,1509..1511,1512..2225)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1551..1572)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1812..1831)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2159..2180
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2426..2560
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2463..2482)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2517..2536
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2578..2625
                     /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)."
     primer_bind     complement(2649..2665)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2649..2665)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2662..2684)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2673..2689
                     /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(2697..2703,2704..2721,2722..2727))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2742..2763
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2880..2897)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3051..3639)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3131..3150)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3810..4601,4602..4670))
                     /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     4433..4452
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4671..4775)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4843..4861
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4899..4921)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5021..5040
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5234..5256
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5248..5265
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5249..5265
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aagcttcctt ctgcaggagc gtacagaacc cagggccctg gcacccgtgc agaccctggc
       61 ccaccccacc tgggcgctca gtgcccaaga gatgtccaca cctaggatgt cccgcggtgg
      121 gtggggggcc cgagagacgg gcaggccggg ggcaggcctg gccatgcggg gccgaaccgg
      181 gcactgccca gcgtggggcg cgggggccac ggcgcgcgcc cccagccccc gggcccagca
      241 ccccaaggcg gccaacgcca aaactctccc tcctcctctt cctcaatctc gctctcgctc
      301 tttttttttt tcgcaaaagg aggggagagg gggtaaaaaa atgctgcact gtgcggcgaa
      361 gccggtgagt gagcggcgcg gggccaatca gcgtgcgccg ttccgaaagt tgccttttat
      421 ggctcgagcg gccgcggcgg cgccctataa aacccagcgg cgcgacgcgc caccaccgcc
      481 gagaccgcgt ccgccccgcg agcacagagc ctcgcctttg ccaccatggc tataacttcg
      541 tatagcatac attatacgaa cggtagtgat aggcctttta gctacatctg ccaatccatc
      601 tcattttcac acacacacac accactttcc ttctggtcag tgggcacatg tccagcctca
      661 agtttatatc accaccccca atgcccaaca cttgtatggc cttgggcggg tcatcccccc
      721 ccccaccccc agtatctgca acctcaagct agcttgggtg cgttggttgt ggataagtag
      781 ctagactcca gcaaccagta acctctgccc tttctcctcc atgacaacca ggtaccggta
      841 gtgactaata gctgatagaa ttccgccccc cccccccccc cctctccctc ccccccccct
      901 aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt
      961 tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg
     1021 acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc
     1081 gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt
     1141 tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta
     1201 taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg
     1261 gaaagagtca aatggctctc ctaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     1321 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     1381 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     1441 cacgatgata ataggatcgc atgcggcgcg ccactagtga tctgctagcg aagcttgccg
     1501 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1561 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1621 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1681 ccaccctcgt gaccaccttc acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1741 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1801 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1861 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1921 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1981 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2041 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2101 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2161 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2221 agtaaagcgg ccgctcgagc gccatcgcta gcggatccac cacctacagc ccagtggggt
     2281 cgctctagag cgacctcgag ggggggctag ataagtaatg atcataatca gccatatcac
     2341 atctgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     2401 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     2461 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2521 tttgtccaaa ctcatcaatg tatcttatca tgtctagatc cgggggtacc gcgtcgagaa
     2581 gttcctattc cgaagttcct attctctaga aagtatagga acttcgtcga cgcgaattcg
     2641 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     2701 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     2761 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2821 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2881 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2941 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3001 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3061 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3121 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3181 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3241 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3301 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3361 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3421 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3481 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3541 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3601 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3661 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3721 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3781 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3841 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3901 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3961 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4021 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4081 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4141 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4201 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4261 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4321 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4381 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4441 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4501 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4561 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4621 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4681 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4741 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4801 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     4861 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     4921 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     4981 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     5041 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     5101 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     5161 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     5221 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctt
//
