LOCUS       sequence-437446-        4271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP reporter construct with double unprocessed direct repeat (35nt)
            at 5' and 3' end, 22nt mCherry-targeting spacer in between DRs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4271)
  TITLE     CRISPR-Cas transgene repressible element
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4271)
  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: 
            "Unpublished"
COMMENT     Sequence Label: 1203F-5'+3'dDR-mCh(22)-GFP-bGHpolyA
FEATURES             Location/Qualifiers
     source          1..4271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1062..1286
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1275..1292
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1315..1332)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(1430..2143,2144..2146,2147..2149))
                     /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(1433..2143,2144..2146,2147..2149))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1475..1496)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1824..1843
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2083..2104
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(2283..2486)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2312..2332)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2487..2866
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3038..3057
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3136..3240
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3241..3309,3310..4101)
                     /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(3459..3478)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctcttcccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcatcctag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtatc gagccccagc tggttctttc cgcctcagaa gccatagagc ccaccgcatc
     1081 cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc
     1141 ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga
     1201 aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac
     1261 agatggctgg caactagaag gcacagtcga ggctgatcag cgggtttaaa ctcaatggtg
     1321 atggtgatga tggttccgtg acatcaaaga aaaccggctt tccaaccgga gttcatgcgc
     1381 ttcaaggtgg ttccgtgaca tcaaagaaaa ccggctttcc aaccaccggt tacttgtaca
     1441 gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat
     1501 cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg
     1561 gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca
     1621 cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct
     1681 tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga
     1741 tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc
     1801 cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc
     1861 gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt
     1921 cggggtagcg gctgaagcac tgcactccat aggtcagggt ggtcacgagg gtgggccagg
     1981 gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat
     2041 cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga
     2101 ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg cggccgcgtt
     2161 ccgtgacatc aaagaaaacc ggctttccaa ccggagttca tgcgcttcaa ggtggttccg
     2221 tgacatcaaa gaaaaccggc tttccaaccg gatctctagc ggatctgacg gttcactaaa
     2281 ccagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg
     2341 gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca
     2401 ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca
     2461 ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac
     2521 tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta
     2581 ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag
     2641 tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc tattggcgtt
     2701 actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca
     2761 ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat
     2821 gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaacg cgtatatctg
     2881 gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt catgcaattg
     2941 tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc agcgacctcc
     3001 aacacacaag cagggagcag atactggctt aactatgcgg catcagagca gattgtactg
     3061 agagtcgacc ctaggggatc gggagatcga tctcccgatc cgtcgacgtc aggtggcact
     3121 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3181 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3241 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3301 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3361 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3421 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3481 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3541 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3601 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3661 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3721 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3781 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3841 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3901 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3961 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4021 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4081 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4141 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4201 accaaaatcc cttaacgtga gttttcgttc cactgagtgt cagaccccgt agaaaagatc
     4261 aaaggatctt c
//
