LOCUS       sequence-11442-e        4650 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 4650)
  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 4650)
  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-TAP
FEATURES             Location/Qualifiers
     source          1..4650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      517..526
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(529..562)
                     /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    876..1448
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    899..1450
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1043..1060)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             1476..1493
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1521..1541
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1548..1613
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    1802..1936
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1839..1858)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1893..1912
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1954..2001
                     /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(2025..2041)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2025..2041)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2038..2060)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2049..2065
                     /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(2073..2079,2080..2097,2098..2103))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2118..2139
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2256..2273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2427..3015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2507..2526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3186..3977,3978..4046))
                     /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     3809..3828
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4047..4151)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4219..4237
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4275..4297)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4397..4416
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4610..4632
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4624..4641
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4625..4641
                     /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 gtcggcgaag
      361 ccggtgagtg agcggcgcgg ggccaatcgc gtgcgccgtt ccgaaagttg ccttttatgg
      421 ctcgagcggc cgcggcggcg ccctataaaa cccagcggcg cgacgcgcca ccaccgccga
      481 gaccgcgtcc gcccgcgagc acagagcctc gcctttgcca ccatggctat aacttcgtat
      541 agcatacatt atacgaacgg tagtgatagg ccttttagct acatctgcca atccatctca
      601 ttttcacaca cacacacacc actttccttc tggtcagtgg gcacatgtcc agcctcaagt
      661 ttatatcacc acccccaatg cccaacactt gtatggcctt gggcgggtca tccccccccc
      721 cacccccagt atctgcaacc tcaagctagc ttgggtgcgt tggttgtgga taagtagcta
      781 gactccagca accagtaacc tctgcccttt ctcctccatg acaaccaggt accggtagtg
      841 actaatagct gatagaattc cgcccccccc ccccccccct ctccctcccc cccccctaac
      901 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc
      961 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg
     1021 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg
     1081 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc
     1141 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa
     1201 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa
     1261 agagtcaaat ggctctccta agcgtattca acaaggggct gaaggatgcc cagaaggtac
     1321 cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga
     1381 ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac
     1441 gatgataata ggatcgcatg cggcgcgcca ctagtcatca ccatcaccat cacgattatg
     1501 atattccaac tactgctagc gagaatttgt attttcaggg tgagctcgac tacaaagacc
     1561 atgacggtga ttataaagat catgacatcg actacaagga tgacgatgac aagtaggata
     1621 tcgctagcgg atccaccacc tacagcccag tggggtcgct ctagagcgac ctcgaggggg
     1681 ggctagataa gtaatgatca taatcagcca tatcacatct gtagaggttt tacttgcttt
     1741 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     1801 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     1861 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     1921 ttatcatgtc tagatccggg ggtaccgcgt cgagaagttc ctattccgaa gttcctattc
     1981 tctagaaagt ataggaactt cgtcgacgcg aattcgtaat catggtcata gctgtttcct
     2041 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     2101 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     2161 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2221 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2281 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2341 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2401 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2461 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2521 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2581 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2641 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2701 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2761 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2821 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     2881 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2941 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3001 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3061 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3121 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3181 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3241 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3301 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3361 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3421 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3481 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3541 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3601 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3661 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3721 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3781 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3841 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3901 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3961 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4021 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4081 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4141 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     4201 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     4261 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     4321 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     4381 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     4441 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     4501 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     4561 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     4621 cgttgtaaaa cgacggccag tgccaagctt
//
