LOCUS       sequence-394844-        4160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Strong EGFP fluorescence recombination plasmid for Matreyek Bxb1(GA)
            landing pad.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4160)
  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 4160)
  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: G413A_BattB(GA)-EGFP
FEATURES             Location/Qualifiers
     source          1..4160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     621..640
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(859..963)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(989..1444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1126..1147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1338..1357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1571..1593
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1585..1602
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1586..1602
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1609..1628
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1609..1627
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1701..1738
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      1763..1772
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1769..1771,1772..1774,1775..2488)
                     /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(1769..1771,1772..1774,1775..2485)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1814..1835)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2075..2094)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2422..2443
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2828..2949
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2865..2884)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2919..2938
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(2958..2976)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2997..3013)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2997..3013)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3010..3032)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3021..3037
                     /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(3045..3051,3052..3069,3070..3075))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3090..3111
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3228..3245)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3399..3987)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3479..3498)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4158..4160,1..789,790..858))
                     /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"
ORIGIN
        1 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
       61 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      121 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
      181 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
      241 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
      301 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
      361 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
      421 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
      481 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
      541 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
      601 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
      661 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      721 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      781 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      841 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      901 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      961 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     1021 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     1081 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     1141 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     1201 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     1261 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     1321 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     1381 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     1441 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     1501 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     1561 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca
     1621 ctatagggcg aattgggtac cgggcccccc ctcgaggtcg acgatgtagg tcacggcatt
     1681 ccggactcag cgctcgagcc ggcttgtcga cgacggcgga ctccgtcgtc aggatcatcc
     1741 gcagattata ccgcaactac acgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     1801 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     1861 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     1921 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     1981 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2041 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2101 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2161 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2221 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     2281 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     2341 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     2401 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     2461 tctcggcatg gacgagctgt acaagtgaag gacggcggca tggacgagct gtacaagtga
     2521 aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc
     2581 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
     2641 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
     2701 agctgtacaa gtagggatcc tgatcataat cagccatacc acatttgtag aggttttact
     2761 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2821 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2881 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2941 tgtatcttag gagatcccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca
     3001 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3061 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3121 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3181 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3241 tcgctgcact cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3301 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3361 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3421 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3481 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3541 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3601 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3661 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3721 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3781 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3841 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3901 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3961 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4021 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4081 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4141 taaacttggt ctgacagtta
//
