LOCUS       sequence-388711-        6427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre reporter for mammalian cells; double-floxxed-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6427)
  AUTHORS   Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F
  TITLE     Programmable protein delivery with a bacterial contractile injection
            system.
  JOURNAL   Nature. 2023 Apr;616(7956):357-364. doi: 10.1038/s41586-023-05870-7.
            Epub 2023 Mar 29.
  PUBMED    36991127
REFERENCE   2  (bases 1 to 6427)
  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: 
            "10.1038/s41586-023-05870-7"; journalName: "Nature"; date: 
            "2023-04"; volume: "616"; issue: "7956"; pages: "357-364"
COMMENT     Sequence Label: pcDNA3-Dio-GFP
FEATURES             Location/Qualifiers
     source          1..6427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(110..129)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        301..680
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        681..884
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     835..855
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     929..948
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        929..947
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(1004..1037)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1088..1121)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1138..1851,1852..1854,1855..1857))
                     /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(1141..1851,1852..1854,1855..1857))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1183..1204)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1532..1551
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1791..1812
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    1874..1907
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1958..1991
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2084..2101)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2090..2314
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2360..2788
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2447..2466)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2657..2678
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2797..2817)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2802..3131
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2982..3117
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3044..3063
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3198..3992
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3252..3271)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3862..3881
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4166..4287
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4203..4222)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4257..4276
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4336..4352)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4336..4352)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4349..4371)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4360..4376
                     /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(4384..4390,4391..4408,4409..4414))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4429..4450
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4567..4584)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4738..5326)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4818..4837)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5497..6288,6289..6357))
                     /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     6120..6139
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6358..6427,1..35))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
       61 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
      121 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
      181 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
      241 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
      301 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      361 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      421 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      481 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      541 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      601 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      661 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      721 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      781 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      841 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      901 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      961 gctagcgttt aaacttaagc ttggtaccga gctcggatcc ggaataactt cgtataggat
     1021 actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag caatatgaaa
     1081 cctcttaata acttcgtata gcatacatta tacgaagtta tttaattaag cggccgctta
     1141 cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac
     1201 catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactgggtgc tcaggtagtg
     1261 gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc
     1321 gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt
     1381 cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg
     1441 ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag
     1501 ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa
     1561 gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt
     1621 catgtggtcg gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt
     1681 gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta
     1741 ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc
     1801 cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatggg
     1861 gcggcgcgcc gcgataactt cgtataaagt atcctatacg aagttatttg ccttaaccca
     1921 gaaattatca ctgttattct ttagaatggt gcaaagaata acttcgtata atgtatgcta
     1981 tacgaagtta tgtttaaacc aatcaacctc tgggaattct gcagatatcc agcacagtgg
     2041 cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     2101 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2161 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2221 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2281 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2341 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2401 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2461 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2521 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2581 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2641 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2701 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2761 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     2821 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     2881 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2941 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3001 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3061 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3121 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     3181 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3241 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3301 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3361 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3421 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3481 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3541 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     3601 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     3661 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     3721 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     3781 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     3841 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     3901 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     3961 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4021 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4081 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4141 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4201 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4261 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4321 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4381 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4441 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4501 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4561 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4621 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4681 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4741 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4801 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4861 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4921 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4981 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5041 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5101 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5161 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5221 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5281 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5341 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5401 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5461 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5521 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5581 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5641 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5701 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5761 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5821 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5881 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5941 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6001 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6061 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6121 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6181 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6241 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6301 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6361 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6421 atttgaa
//
