LOCUS       sequence-437434-        4122 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP reporter construct with single unprocessed direct repeat (35nt)
            at 5' end.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4122)
  TITLE     CRISPR-Cas transgene repressible element
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4122)
  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            "Unpublished"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: 1203A-CMV-5'DR-EGFP-bGHpolyA
FEATURES             Location/Qualifiers
     source          1..4122
                     /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(1338..2051,2052..2054,2055..2057))
                     /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(1341..2051,2052..2054,2055..2057))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1383..1404)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1732..1751
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1991..2012
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(2134..2337)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2163..2183)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2338..2717
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2889..2908
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2987..3091
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3092..3160,3161..3952)
                     /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(3310..3329)
                     /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 tgaccggtta cttgtacagc tcgtccatgc cgagagtgat cccggcggcg
     1381 gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt gctcagggcg
     1441 gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg
     1501 ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg
     1561 aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt gtggctgttg
     1621 tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc
     1681 agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag
     1741 ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg catggcggac
     1801 ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg cactccatag
     1861 gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac
     1921 ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac gctgaacttg
     1981 tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc
     2041 tcgcccttgc tcaccatgcg gccgcgttcc gtgacatcaa agaaaaccgg ctttccaacc
     2101 ggatctctag cggatctgac ggttcactaa accagctctg cttatataga cctcccaccg
     2161 tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc
     2221 ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga gacttggaaa
     2281 tccccgtgag tcaaaccgct atccacgccc attgatgtac tgccaaaacc gcatcaccat
     2341 ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat
     2401 gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact
     2461 tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca
     2521 ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt
     2581 caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac
     2641 gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact attaataact
     2701 agtcaataat caatgtcaac gcgtatatct ggcccgtaca tcgcgaagca gcgcaaaacg
     2761 cctaacccta agcagattct tcatgcaatt gtcggtcaag ccttgccttg ttgtagctta
     2821 aattttgctc gcgcactact cagcgacctc caacacacaa gcagggagca gatactggct
     2881 taactatgcg gcatcagagc agattgtact gagagtcgac cctaggggat cgggagatcg
     2941 atctcccgat ccgtcgacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3001 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3061 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3121 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3181 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3241 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3301 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3361 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3421 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3481 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3541 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3601 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3661 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3721 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3781 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3841 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3901 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3961 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4021 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4081 ccactgagtg tcagaccccg tagaaaagat caaaggatct tc
//
