LOCUS       sequence-466774-        4087 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4087)
  TITLE     CTRL Enables Gene-specific RNA Regulation Using CRISPR-Cas7-11
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4087)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: 1162BX-CMV-EGFP-bGHpolyA
FEATURES             Location/Qualifiers
     source          1..4087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     528..547
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(766..870)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(949..968)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1140..1519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1520..1723
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1674..1694
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1765..1767,1768..1770,1771..2484)
                     /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(1765..1767,1768..1770,1771..2481)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1810..1831)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2071..2090)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2418..2439
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2490..2507
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2530..2547)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2536..2760
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2831..2847)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2831..2847)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2844..2866)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2855..2871
                     /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(2879..2885,2886..2903,2904..2909))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2924..2945
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3062..3079)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3233..3821)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3313..3332)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3992..4087,1..696,697..765))
                     /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"
ORIGIN
        1 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
       61 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
      121 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
      181 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
      241 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
      301 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
      361 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
      421 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
      481 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
      541 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
      601 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
      661 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
      721 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
      781 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
      841 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
      901 cgacggatcg ggagatcgat ctcccgatcc cctagggtcg actctcagta caatctgctc
      961 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     1021 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     1081 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
     1141 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     1201 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     1261 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     1321 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     1381 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     1441 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     1501 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     1561 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     1621 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     1681 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
     1741 gatccgctag agatccgcgg ccgcatggtg agcaagggcg aggagctgtt caccggggtg
     1801 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1861 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1921 aagctgcccg tgccctggcc caccctcgtg accaccctga cctatggagt gcagtgcttc
     1981 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     2041 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     2101 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2161 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     2221 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     2281 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     2341 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     2401 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     2461 ggcatggacg agctgtacaa gtaaccggtc atcatcacca tcaccattga gtttaaaccc
     2521 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2581 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2641 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2701 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2761 cttctgaggc ggaaagaacc agctggggct cgataccgtc gacctctagc tagagcttgg
     2821 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     2881 acatacgagc cggaagcata aagtgtaaag cctaggatgc ctaatgagtg agctaactca
     2941 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3001 attaatgaat cggccaacgc gcgggaagag gcggtttgcg tattgggcgc tcttccgctt
     3061 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3121 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3181 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3241 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3301 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3361 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3421 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3481 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3541 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3601 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3661 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3721 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3781 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3841 ctacggggtc tgacactcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3901 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3961 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4021 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4081 ctacgat
//
