LOCUS       sequence-437451-        4191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP reporter construct with double processed direct repeats (15nt) 
            and a non-target spacer in between at 5' and 3' end.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4191)
  TITLE     CRISPR-Cas transgene repressible element
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4191)
  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            "Unpublished"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: 1203S-5'+3'dmDR-NT(22)-GFP-bGHpolyA
FEATURES             Location/Qualifiers
     source          1..4191
                     /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(1390..2103,2104..2106,2107..2109))
                     /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(1393..2103,2104..2106,2107..2109))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1435..1456)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1784..1803
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2043..2064
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(2203..2406)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2232..2252)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2407..2786
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2958..2977
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3056..3160
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3161..3229,3230..4021)
                     /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(3379..3398)
                     /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 acatcaaaaa tacaagggct accaggtagg ttccgtgaca
     1381 tcaaaccggt tacttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa
     1441 ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactgggt
     1501 gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg
     1561 gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct tgaagttcac
     1621 cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta
     1681 ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat
     1741 gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc
     1801 gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa
     1861 gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac tgcactccat aggtcagggt
     1921 ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt
     1981 cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt
     2041 tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt
     2101 gctcaccatg cggccgcgtt ccgtgacatc aaaaatacaa gggctaccag gtaggttccg
     2161 tgacatcaag gatctctagc ggatctgacg gttcactaaa ccagctctgc ttatatagac
     2221 ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt
     2281 tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag
     2341 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg
     2401 catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca
     2461 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     2521 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     2581 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     2641 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     2701 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     2761 ttaataacta gtcaataatc aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag
     2821 cgcaaaacgc ctaaccctaa gcagattctt catgcaattg tcggtcaagc cttgccttgt
     2881 tgtagcttaa attttgctcg cgcactactc agcgacctcc aacacacaag cagggagcag
     2941 atactggctt aactatgcgg catcagagca gattgtactg agagtcgacc ctaggggatc
     3001 gggagatcga tctcccgatc cgtcgacgtc aggtggcact tttcggggaa atgtgcgcgg
     3061 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3121 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3181 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3241 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3301 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3361 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3421 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3481 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3541 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3601 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3661 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3721 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3781 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3841 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     3901 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3961 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4021 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4081 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4141 gttttcgttc cactgagtgt cagaccccgt agaaaagatc aaaggatctt c
//
