LOCUS       sequence-335069-        4160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Strong EGFP fluorescence from Bxb1 landing pad.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4160)
  AUTHORS   Shukla N, Roelle SM, Suzart VG, Bruchez AM, Matreyek KA
  TITLE     Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2
            spike mediated infection.
  JOURNAL   PLoS Pathog. 2021 Jul 16;17(7):e1009715. doi: 
            10.1371/journal.ppat.1009715. eCollection 2021 Jul.
  PUBMED    34270613
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: "PLoS 
            Pathog."; date: "2021-07-16"; volume: "17(7):e1009715. doi"; pages: 
            " 10.1371/journal.ppat.1009715. eCollection 2021 Jul"
COMMENT     Sequence Label: AttB _EGFP
FEATURES             Location/Qualifiers
     source          1..4160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    520..541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(556..561,562..579,580..586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    594..610
                     /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)."
     primer_bind     599..621
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     618..634
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     618..634
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        655..673
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    682..803
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(693..712)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     747..766
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(1143..1856,1857..1859,1860..1862))
                     /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             complement(join(1146..1856,1857..1859,1860..1862))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1188..1209)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1537..1556
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1796..1817
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1859..1868
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1893..1930
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     primer_bind     complement(2003..2022)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2004..2022)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2029..2046)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2029..2045)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2038..2060)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2187..2642
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2274..2293)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2484..2505
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2668..2772
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2773..2841,2842..3633)
                     /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     complement(2991..3010)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3804..4160,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agtgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      541 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      601 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc tcgaaattaa
      661 ccctcactaa aggggatctc ctaagataca ttgatgagtt tggacaaacc acaactagaa
      721 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
      781 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
      841 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg
      901 attatgatca ggatccctac ttgtacagct cgtccatgcc gccggtggag tggcggccct
      961 cggcgcgttc gtactgttcc acgatggtgt agtcctcgtt gtgggaggtg atgtccaact
     1021 tgatgttgac gttgtaggcg ccgggcagct gcacgggctt cttggccttg taggtggtct
     1081 tgacctcagc gtcgtagtgg ccgccgtcct tcacttgtac agctcgtcca tgccgccgtc
     1141 cttcacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     1201 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg
     1261 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     1321 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg
     1381 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
     1441 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
     1501 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
     1561 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
     1621 tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg
     1681 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
     1741 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
     1801 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
     1861 atggtggcgt gtagttgcgg tataatctgc ggatgatcct gacgacggag accgccgtcg
     1921 tcgacaagcc ggctcgagcg ctgagtccgg aatgccgtga cctacatcgt cgacctcgag
     1981 ggggggcccg gtacccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt
     2041 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     2101 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     2161 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2221 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2281 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2341 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2401 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2461 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2521 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2581 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2641 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2701 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2761 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2821 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2881 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2941 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3001 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3061 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3121 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3181 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3241 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     3301 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     3361 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3421 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3481 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3541 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3601 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3661 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3721 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3781 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3841 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3901 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     3961 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4021 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4081 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4141 cagcccagct tggagcgaac
//
