LOCUS       sequence-62684-e        5385 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5385)
  AUTHORS   Yang YS, Hughes TE
  TITLE     Cre stoplight: a red/green fluorescent reporter of Cre recombinase 
            expression in living cells.
  JOURNAL   Biotechniques. 2001 Nov;31(5):1036, 1038, 1040-1.
  PUBMED    11730010
REFERENCE   2  (bases 1 to 5385)
  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: 
            "Biotechniques."; date: "2001-11"; volume: "31(5)"; pages: "1036, 
            1038, 1040-1"
COMMENT     Sequence Label: Cre Stoplight
FEATURES             Location/Qualifiers
     source          1..5385
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..209
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     160..180
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     254..273
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        254..272
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(292..325)
                     /label=loxH
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021). The loxH variant 
                     was optimized for expression constructs but remains 
                     compatible with loxP."
     regulatory      355..364
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(361..363,364..366,367..1041)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="MVRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
                     HLFL"
     primer_bind     complement(544..564)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     762..781
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     956..979
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1114..1131
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1148..1165)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1154..1361
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1419..1437)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1433..1480
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     protein_bind    complement(1526..1559)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1589..1598
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1595..1597,1598..1600,1601..2314)
                     /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(1595..1597,1598..1600,1601..2311)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1640..1661)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1901..1920)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2248..2269
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2452..2573
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2489..2508)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2543..2562
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2622..2638)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2622..2638)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2635..2657)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2646..2662
                     /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(2670..2676,2677..2694,2695..2700))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2715..2736
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2853..2870)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3024..3609)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3104..3123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3780..4571,4572..4640))
                     /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     4403..4422
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4641..4745)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4820..4839)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(5011..5385,1..5)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
       61 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      121 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      181 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
      241 ggcttatcga aattaatacg actcactata gggagaccca agctggctag aattacctca
      301 tatagcatac attatacgaa gttatcggag gaattggctc gaggaattgc ccttgccacc
      361 atggtgcgct cctccaagaa cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag
      421 ggcaccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
      481 ggccacaaca ccgtgaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac
      541 atcctgtccc cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc
      601 cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
      661 gaggacggcg gcgtggtgac cgtgacccaa gactcctccc tgcaggacgg ctgcttcatc
      721 tacaaggtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat gcagaagaag
      781 accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc
      841 gagatccaca aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagtcc
      901 atctacatgg ccaagaagcc cgtgcagctg cccggctact actacgtgga ctccaagctg
      961 gacatcacct cccacaacga ggactacacc atcgtggagc agtacgagcg caccgagggc
     1021 cgccaccacc tgttcctgta gcggcctcga ctaagggcaa ttcgggagct cggtaagcct
     1081 atccctaacc ctctcctcgg tctcgattct agccatcatc accatcacca ttgaagctcg
     1141 ctatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1201 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     1261 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     1321 aagggggagg attgggaaga caatagcagg catgctgggg attctagaag atccggctgc
     1381 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     1441 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     1501 cggatatccc agcggccgcg gtaccataac ttcgtatagc atacattata cgaagttatg
     1561 tttcggaatt cggcttcgag atctggtcgc caccatggtg agcaagggcg aggagctgtt
     1621 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1681 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1741 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1801 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1861 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1921 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1981 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     2041 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     2101 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2161 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     2221 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2281 gatcactctc ggcatggacg agctgtacaa gtaaagcggc cagatctcta gaagccgaat
     2341 tcgattccac ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     2401 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     2461 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     2521 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     2581 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     2641 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     2701 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     2761 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     2821 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     2881 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     2941 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3001 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3061 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3121 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3181 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3241 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3301 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3361 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3421 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3481 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3541 caaaccaccg ctggtagcgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3601 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3661 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3721 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3781 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3841 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3901 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3961 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4021 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4081 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4141 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4201 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4261 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4321 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4381 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4441 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4501 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4561 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4621 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4681 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4741 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     4801 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     4861 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     4921 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     4981 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
     5041 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     5101 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     5161 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     5221 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     5281 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     5341 actttcctac ttggcagtac atctacgtat tagtcatcgc tatta
//
