LOCUS       Exported                6651 bp DNA     circular SYN 05-JUL-2022
DEFINITION  attB1-attB2.
ACCESSION   .
VERSION     .
KEYWORDS    pAFMW_Nbr Exo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6651)
  AUTHORS   Ivica
  TITLE     Direct Submission
  JOURNAL   Exported Jul 5, 2022 from SnapGene Viewer 6.0.6
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    38..880
                     /label=SV40 sti/polyA
                     /label=SV40\sti/polyA
                     /note="/vntifkey=25"
                     /note="color: #a6acb3"
     intron          106..171
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
                     /note="color: #------"
     CDS             301..321
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     polyA_signal    746..880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     CDS             977..1837
                     /codon_start=1
                     /label=ampR
                     /note="/vntifkey=4"
                     /note="color: #666699"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2008..2596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     promoter        2868..5440
                     /label=Actin5C
                     /note="/vntifkey=29"
                     /note="color: #ffffff"
     promoter        2918..5427
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
                     /note="color: #ffffff; direction: RIGHT"
     regulatory      5536..5545
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             5542..5883
                     /codon_start=1
                     /label=3xFLAG-6xMyc
                     /note="/vntifkey=4"
                     /note="color: #993366"
                     /translation="MDYKDHDGDYKDHDIDYKDDDDKHRLSSATMEQKLISEEDLNEME
                     QKLISEEDLNEMEQKLISEEDLNEMEQKLISEEDLNEMEQKLISEEDLNEMESLGDLTM
                     EQKLISEEDL"
     CDS             5545..5610
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /note="color: #cc99b2
                     Cleavage site after base 5610"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      5626..5635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             5635..5664
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             5674..5703
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             5713..5742
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             5752..5781
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             5791..5820
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             5854..5883
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     protein_bind    5896..5920
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
                     /note="color: #31849b; direction: RIGHT"
     misc_feature    5897..5917
                     /label=attB1
                     /note="/vntifkey=21"
                     /note="color: #a6acb3"
     primer_bind     complement(5926..5940)
                     /label=G4_PCR5_rev
                     /note="color: black; sequence: 
                     TGGGAAAAAAGGGCTTCACGggtgaagggggcggc; added: 2019-09-13"
     primer_bind     complement(5926..5940)
                     /label=G3_PCR2_rev
                     /note="color: black; sequence: 
                     TGCAAGACAGCCTGGCAATCggtgaagggggcggc; added: 2019-09-12"
     protein_bind    5932..5936
                     /label=TOPO binding site
                     /label=TOPO\binding\site
                     /note="/invitrogen=2470000"
                     /note="/vntifkey=31"
                     /note="color: #31849b"
     protein_bind    5937..5940
                     /label=directional TOPO overhang
                     /label=directional\TOPO\overhang
                     /note="/invitrogen=2410000"
                     /note="/vntifkey=31"
                     /note="color: #31849b"
     misc_feature    5941..6624
                     /label=Nbr-Exo CM (CtermDom)
                     /label=Nbr-Exo\(CtermDom)
                     /note="/vntifkey=21"
                     /note="color: #33cccc"
     primer_bind     5941..5961
                     /label=G4_PCR5_FWD
                     /note="color: black; sequence: 
                     CcGCCGGcTCTGGGGAGTTtatgtacctgaccatggacctg; added: 
                     2019-09-13"
     primer_bind     5941..5961
                     /label=G3_PCR2_fwd
                     /note="color: black; sequence: 
                     CcGCCGGcTCTGGGGAGTTTatgtacctgaccatggacctg; added: 
                     2019-09-13"
     misc_feature    6049..6051
                     /label=D435A
                     /note="D435A: GAT=>GCC"
                     /note="color: #ff9900"
     misc_feature    6055..6057
                     /label=E437A
                     /note="E437A
                     GAA=>GCC"
                     /note="color: #ff9900"
     primer_bind     complement(6604..6621)
                     /label=G1_PCR0_rev
                     /note="color: black; sequence: cttaacatgggcaccccg; added: 
                     2019-09-11"
     primer_bind     complement(6605..6624)
                     /label=P16_NbrCatdom_rev
                     /note="color: black; sequence: tcacttaacatgggcacccc; added:
                     2019-10-09"
     primer_bind     6625..6640
                     /label=G1_PCR0_fwd
                     /note="color: black; sequence: aagggtgggcgcgccg; added: 
                     2019-09-11"
     protein_bind    complement(6625..6629)
                     /label=TOPO binding site
                     /label=TOPO\binding\site
                     /note="/invitrogen=2460000"
                     /note="/vntifkey=31"
                     /note="color: #31849b; direction: LEFT"
     protein_bind    complement(join(6641..6651,1..14))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
                     /note="color: #31849b; direction: LEFT"
     misc_feature    complement(join(6644..6651,1..13))
                     /label=attB2
                     /note="/vntifkey=21"
                     /note="color: #a6acb3; direction: LEFT"
ORIGIN
        1 ttgtacaaag tggtgacgta agctagcagg atctttgtga aggaacctta cttctgtggt
       61 gtgacataat tggacaaact acctacagag atttaaagct ctaaggtaaa tataaaattt
      121 ttaagtgtat aatgtgttaa actactgatt ctaattgttt gtgtatttta gattccaacc
      181 tatggaactg atgaatggga gcagtggtgg aatgccttta atgaggaaaa cctgttttgc
      241 tcagaagaaa tgccatctag tgatgatgag gctactgctg actctcaaca ttctactcct
      301 ccaaaaaaga agagaaaggt agaagacccc aaggactttc cttcagaatt gctaagtttt
      361 ttgagtcatg ctgtgtttag taatagaact cttgcttgct ttgctattta caccacaaag
      421 gaaaaagctg cactgctata caagaaaatt atggaaaaat attctgtaac ctttataagt
      481 aggcataaca gttataatca taacatactg ttttttctta ctccacacag gcatagagtg
      541 tctgctatta ataactatgc tcaaaaattg tgtaccttta gctttttaat ttgtaaaggg
      601 gttaataagg aatatttgat gtatagtgcc ttgactagag atcataatca gccataccac
      661 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
      721 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
      781 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
      841 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc ccgtttaaac tacgcgtaat
      901 tcaaacaggg ttctggcgtc gttctcgtac tgttttcccc aggccagtgc tttagcgtta
      961 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1021 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1081 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1141 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1201 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1261 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     1321 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     1381 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     1441 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     1501 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     1561 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     1621 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     1681 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     1741 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     1801 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1861 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1921 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1981 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2041 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2101 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2161 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2221 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2281 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2341 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2401 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2461 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2521 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2581 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     2641 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2701 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     2761 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     2821 tcattaatgc aggcaactcg tgaaaggtag gcggatcagc ggccgcagca tgcaattcta
     2881 tattctaaaa acacaaatga tacttctaaa aaaaaatcat gaatggcatc aactctgaat
     2941 caaatctttg cagatgcacc tacttctcat ttccactgtc acatcatttt tccagatctc
     3001 gctgcctgtt atgtggccca caaaccaaga cacgttttat ggccattaaa gctggctgat
     3061 cgtcgccaaa caccaaatac ataatgaata tgtacacatt cgagaaagaa gcgatcaaag
     3121 aagcgtcttc gggcggagta ggagaatgcg gaggagaagg agaacgagct gatctagtat
     3181 ctctccacaa tccaatgcca actgaccaac tggccatatt cggagcaatt tgaagccaat
     3241 ttccatcgcc tggcgatcgc tccattcttg gctatatgtt tttcaccgtt acccggggcc
     3301 attttcaaag actcgtcggc aagataagat tgtgtcactc gctgtctctc ttcatttgtc
     3361 gaagaatgct gaggaatttc gcgatgacgt cggcgagtat tttgaagaat gagaataatt
     3421 tgtatttata cgaaaatcag ttagtggaat tttctacaaa aacatgttat ctatagataa
     3481 ttttgttgca aaatatgttg actatgacaa agattgtatg tatatacctt taatgtattc
     3541 tcattttctt atgtatttat aatggcaatg atgatactga tgatatttta agatgatgcc
     3601 agaccaaaag gcttgaattt ctgcgtcttt tgccgaacgc agtgcatgtg caattgttgt
     3661 tttttggaat attcaatttt cggactgtcc gctttgattt cagtttcttg gcttattcaa
     3721 aaagcaaagt aaagccaaaa aagcgagatg gcaataccaa atgcggcaaa acggtagtgg
     3781 aaggaaaggg gtgcggggca gcggaaggaa gggtggggcg gggcgtggcg gggtctgtgg
     3841 ctgggcgcga cgtcaccgac gttggagcca ctcctttgac catgtgtgcg tgtgtgtatt
     3901 attcgtgtct cgccactcgc cggttgtttt tttcttttta tgctgcgctc tctctagcgc
     3961 catctcgctt acgcatgctc aacgcaccgc atgttgccgt ttccttttat gcgtcatttt
     4021 ggctcgaaat aggcaattat ttaaacaaag attagtcaac gaaaacgcta aaataaataa
     4081 gtctacaata tggttactta ttgccatgtg tgtgcagcca acgatagcaa caaaagcaac
     4141 aacacaggtg gctttccctc tttcactttt tgtttgcaag ccgcgtgcga gcaagacggc
     4201 acgaccggca aacgcaatta cgctgacaaa gagcagacga agttttggcg aaaaacatca
     4261 aggcgcctga tacgaatgca tttgcaataa caattgcgat atttaatatt gtttatgaag
     4321 ctgtttgact tcaaaacaca caaaaaaaaa aataaaacaa attatttgaa agagaattag
     4381 gaatcggacg cttatcgtta gggtaacaac aagaaatgct tactgagtca cagcctctgg
     4441 aaaactgccg caagccagag agagagagaa aaagagggag agcagcttag accgcatgtg
     4501 cttgtgtgtg aggcgtctct ctcttcgtct ctgttgcgca aacgcataga ctgcactgag
     4561 aaaatcgatt acctattttt tatgaatgaa tatttgcact attactattc aaaactatta
     4621 agatagcaat cacattcaat agccaaatac tataccacct gagcgatgca acgaaatgat
     4681 caatttgagc aaaaatgctg catatttagg acggcatcat tatagaaatg cttcttgctg
     4741 tgtacttttc tctcgtctgg cagctgtttc gccgttattg ttaaaaccgg cttaagttag
     4801 gtgtgttttc tacgactagt gaatgcccta ctagaagatg tgtgttgcac aaaatgtccc
     4861 tggaataacc aatttgaagt gcagatagca gtaaacgtaa gctaatatga atattattta
     4921 actgtaatgt tttaatatcg ctggacatta ctaataaacc cactataaac acatgtacat
     4981 atgtatgttt tggcatacaa tgagtagttg gggaaaaaat gtgtaaaagc accgtgacca
     5041 tcacagcata aagataacca gctgaagtat cgaatatgag taacccccaa attgaatcac
     5101 atgccgcaac tgataggacc catggaagta cactcttcat ggcgatatac aagacacaca
     5161 caagcacgaa cacccagttg cggaggaaat tctccgtaaa tgaaaaccca atcggcgaac
     5221 aattcatacc catatatggt aaaagttttg aacgcgactt gagagcggag agcattgcgg
     5281 ctgataaggt tttagcgcta agcgggcttt ataaaacggg ctgcgggacc agttttcata
     5341 tcactaccgt ttgagttctt gtgctgtgtg gatactcctc ccgacacaaa gccgctccat
     5401 cagccagcag tcgtctaatc cagagacccc ggatccagat atcgaggcct gtctagagaa
     5461 gcttgttcga atctcgagtg cgcgcttccg gaggtataca cctaggcggt accactgcag
     5521 tgaattcgga gctccgccac catggactac aaagaccatg acggtgatta taaagatcat
     5581 gacatcgatt acaaggatga cgatgacaag caccggttga gctccgccac catggagcaa
     5641 aagctcattt ctgaagagga cttgaatgaa atggagcaaa agctcatttc tgaagaggac
     5701 ttgaatgaaa tggagcaaaa gctcatttct gaagaggact tgaatgaaat ggagcaaaag
     5761 ctcatttctg aagaggactt gaatgaaatg gagcaaaagc tcatttctga agaggacttg
     5821 aatgaaatgg agagcttggg cgacctcacc atggagcaaa agctcatttc tgaagaggac
     5881 ttgaatcacc ggtatacaag tttgtacaaa aaagcaggct ccgcggccgc ccccttcacc
     5941 atgtacctga ccatggacct gcccgacgag tgccttatca ttgtgaacaa ggctgatgag
     6001 ttcgatcgta tgttatacca tctgcagcag gagtgcgtca tatacctggc ctctgcctgg
     6061 atgcagagcg tgtgcggaga caatcagctg tgcgtgctac agatcgccac cggccacaat
     6121 gtctacctga tcgattgcct ggcgcgggag agcttacgct cggaacactg gcgcttgctg
     6181 ggcgcgaata tctttaataa cgtgaacatc cgtaaggtgg ggttctctat ggtcagtgat
     6241 ctcagtgtat tgcagcggtc actgcccctg caactgcgcc ttcaaatgcc tcaccactac
     6301 ttggaccttc gcaatctctg gttggagtta aaaaagcagc gcttcggcgt tgaacttccc
     6361 ttcggcaatg ttaaccgggc aggagacgcc cttacggatc tctcattggc atgccttggc
     6421 aagaaactga acaaatcaaa ccagtgctca aactgggcca atagaccgct gcgtcgcgag
     6481 cagattcttt atgcagccat cgatgcgcgg tgtttgatgc tgatctacaa cacgctcatt
     6541 gaacgcgtgt ccttcataca agcggttatc gaaaagagca tcgccagcaa taactttctc
     6601 aggcggggtg cccatgttaa gtgaaagggt gggcgcgccg acccagcttt c
//
