LOCUS       sequence-394841-        6067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Strong EGFP fluorescence recombination plasmid for Matreyek Bxb1(GA)
            landing pad in dual landing pad cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6067)
  AUTHORS   Roelle SM, Kamath ND, Matreyek KA
  TITLE     Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase 
            Sites for the Functional Characterization of Protein Variants.
  JOURNAL   ACS Synth Biol. 2023 Nov 3. doi: 10.1021/acssynbio.3c00355.
  PUBMED    37922210
REFERENCE   2  (bases 1 to 6067)
  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: "ACS Synth 
            Biol. 2023 Nov 3. doi: 10.1021/acssynbio.3c00355."
COMMENT     Sequence Label: 
            G1052C_BattB(GA)_EGFP_IRES-Spycatcher-sfmCherry3C(1-10)-P2A-shBleR
FEATURES             Location/Qualifiers
     source          1..6067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(105..209)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(235..690)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(372..393)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     584..603
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     817..839
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     832..848
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     855..874
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        855..873
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    947..984
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      1009..1018
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1015..1017,1018..1020,1021..1734)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1015..1017,1018..1020,1021..1731)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1060..1081)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1321..1340)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1668..1689
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1856..2430,2431..2433,2434..2442)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2023..2040)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2250..2269
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2446..2784
                     /codon_start=1
                     /product="improved SpyCatcher variant that shows rapid 
                     reaction with SpyTag002 (Keeble et al., 2017)"
                     /label=SpyCatcher002
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     EATKGDAHT"
     CDS             3433..3489
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3490..3864
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3981..4102
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4018..4037)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4072..4091
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(4111..4129)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4150..4166)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4150..4166)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4163..4185)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4174..4190
                     /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(4198..4204,4205..4222,4223..4228))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4243..4264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4381..4398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4552..5140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4632..4651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5311..6067,1..35,36..104))
                     /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     5934..5953
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
       61 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
      121 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
      181 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
      241 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
      301 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
      361 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
      421 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
      481 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
      541 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
      601 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
      661 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
      721 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
      781 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
      841 cggccagtga attgtaatac gactcactat agggcgaatt gggtaccggg ccccccctcg
      901 aggtcgacga tgtaggtcac ggcattccgg actcagcgct cgagccggct tgtcgacgac
      961 ggcggactcc gtcgtcagga tcatccgcag attataccgc aactacacgc caccatggtg
     1021 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1081 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1141 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1201 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1261 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1321 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1381 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1441 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1501 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1561 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1621 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1681 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtagtaactt
     1741 aagaattcac cggtctgacc tccttctccc tctcttcagg tctgcagtcg acggtaccgc
     1801 gggcccggga tccaccggat ctagataact gatcactgtg gaatcagaga gactgcccct
     1861 ctccctctcc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
     1921 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac
     1981 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
     2041 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa
     2101 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
     2161 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg
     2221 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
     2281 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat
     2341 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg
     2401 tggttttcct ttgaaaaaca cgatgataat atggccacaa ccatggtaac caccttatca
     2461 ggtttatcag gtgagcaagg tccgtccggt gatatgacaa ctgaagaaga tagtgctacc
     2521 catattaaat tctcaaaacg tgatgaggac ggccgtgagt tagctggtgc aactatggag
     2581 ttgcgtgatt catctggtaa aactattagt acatggattt cagatggaca tgtgaaggat
     2641 ttctacctgt atccaggaaa atatacattt gtcgaaaccg cagcaccaga cggttatgag
     2701 gtagcaactg ctattacctt tacagttaat gagcaaggtc aggttactgt aaatggcgaa
     2761 gcaactaaag gtgacgctca tactggcggt ggctctggta ccatggagga ggacaacatg
     2821 gccatcatca aggagttcat gagattcaag gtgcacatgg agggcagcgt gaacggccac
     2881 gagttcgaga tcgagggcga gggcgagggc cacccctacg agggcaccca gaccgccagg
     2941 ctgaaggtga ccaagggcga ccccctgccc ttcgcctggg acatcctgag cccccagttc
     3001 atgtacggca gcaaggccta cgtgaagcac cccgccgaca tccccgacta cctgaagctg
     3061 agcttccccg agggcttcac ctgggagaga gtgatgaact tcgaggacgg cggcgtggtg
     3121 gccgtgaccc aggacagcag cctgcaggac ggccagttca tctacaaggt gaagctgctg
     3181 ggcatcaact tccccagcga cggccccgtg atgcagaaga agaccatggg ctgggaggcc
     3241 agcaccgaga gaatgtaccc cgaggacggc gccctgaagg gcgagatcaa ccagagactg
     3301 aagctgaagg acggcggtca ctacgacgcc gaggtgaaga ccacctacag ggccaagaag
     3361 cccgtgcagc tgcccggcgc ctacgacgtg gacatcaagc tggacatcac cagccacaac
     3421 gaggacagcg gagctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac
     3481 cctggaccta tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc
     3541 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac
     3601 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg
     3661 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag
     3721 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc
     3781 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac
     3841 ttcgtggccg aggagcagga ctaataggga tcctgatcat aatcagccat accacatttg
     3901 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     3961 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     4021 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4081 ccaaactcat caatgtatct taggagatcc cctttagtga gggttaattt cgagcttggc
     4141 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4201 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4261 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4321 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4381 ctcgctcact gactcgctgc actcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4441 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4501 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4561 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4621 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4681 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4741 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4801 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4861 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4921 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4981 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5041 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5101 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5161 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5221 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5281 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5341 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5401 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5461 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5521 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5581 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5641 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5701 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5761 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5821 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5881 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5941 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6001 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6061 ctgatct
//
