LOCUS       sequence-82389-e        6206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for producing PhiC31 attB-containing minicircle in vitro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6206)
  AUTHORS   Tasic B, Hippenmeyer S, Wang C, Gamboa M, Zong H, Chen-Tsai Y, Luo L
  TITLE     Site-specific integrase-mediated transgenesis in mice via pronuclear
            injection.
  JOURNAL   Proc Natl Acad Sci U S A. 2011 May 10;108(19):7902-7. doi: 
            10.1073/pnas.1019507108. Epub 2011 Apr 4.
  PUBMED    21464299
REFERENCE   2  (bases 1 to 6206)
  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: 
            "10.1073/pnas.1019507108"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2011-05-10- 10"; volume: "108"; issue: "19"; pages: "7902-7"
COMMENT     Sequence Label: pBT346_pLambda-attB-pCA-GFP-pA
FEATURES             Location/Qualifiers
     source          1..6206
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    689..788
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    816..885
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     enhancer        1191..1494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1496..1773
                     /label=chicken beta-actin promoter
     intron          1775..2792
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2716..2738
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2800..2819
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2870..2879
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2876..2878,2879..2881,2882..3595)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2921..2942)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3182..3201)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3529..3550
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3717..3838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3754..3773)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3808..3827
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    3878..4002
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(4021..4039)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4060..4076)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4060..4076)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4073..4095)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4084..4100
                     /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(4108..4114,4115..4132,4133..4138))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4153..4174
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4291..4308)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4462..5050)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4542..4561)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5221..6012,6013..6081))
                     /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     5844..5863
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6082..6186)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtaccag
      661 ttacgctagg gataacaggg taatatagca aataatgatt ttattttgac tgatagtgac
      721 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      781 agcaggctct cgaggtcgac gatgtaggtc acggtctcga agccgcggtg cgggtgccag
      841 ggcgtgccct tgggctcccc gggcgcgtac tccacctcac ccatctggtc catcatgatg
      901 aacgggtcga ggtggcggta gttgatcccg gcgaacgcgc ggcgcaccgg gaagccctcg
      961 ccctcgaaac cgctgggcgc ggtggtcacg gtgagcacgg gacgtgcgac ggcgtcggcg
     1021 ggtgcggata cgcggggcag cgtcagcggg ttctcgacgg tcacggcggg catgtcgacg
     1081 gtatcgataa gcttccctta attaaccgtt taaacaattc tgcaggaatc tagttattaa
     1141 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     1201 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     1261 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     1321 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     1381 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     1441 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag
     1501 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg
     1561 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc
     1621 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
     1681 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     1741 cggccctata aaaagcgaag cgcgcggcgg gcggggagtc gctgcgacgc tgccttcgcc
     1801 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
     1861 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt
     1921 aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc
     1981 ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg
     2041 cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg
     2101 cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg
     2161 gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg
     2221 tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg
     2281 gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg
     2341 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct
     2401 cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca
     2461 gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct
     2521 gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc
     2581 ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc
     2641 gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg
     2701 gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta
     2761 accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc
     2821 tgtctcatca ttttggcaaa gaattgattt gataccgcgg gcccgggccg ccaccatggt
     2881 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2941 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     3001 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     3061 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     3121 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     3181 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     3241 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     3301 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     3361 caaggcgaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgccgacca
     3421 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     3481 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3541 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctttaca agtaagaatt
     3601 ctagataact gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     3661 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     3721 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3781 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatt
     3841 taaattgggc gcgcccaact agttctagag cggccgcaca agtttgtaca aaaaagctga
     3901 acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca
     3961 gactacataa tactgtaaaa cacaacatat ccagtcacta tggagctcca gcttttgttc
     4021 cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4081 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4141 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4201 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4261 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4321 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4381 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4441 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4501 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4561 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4621 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4681 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4741 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4801 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4861 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     4921 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4981 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5041 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5101 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5161 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5221 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5281 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5341 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5401 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5461 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5521 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5581 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5641 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5701 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5761 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5821 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5881 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5941 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6001 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6061 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6121 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6181 tccgcgcaca tttccccgaa aagtgc
//
