LOCUS       sequence-447435-        4763 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter plasmid for genetic code expansion which 
            consists of a modified GFP with a Amber stop codon on position Y39.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4763)
  TITLE     Lemke Lab Plasmids 2024
REFERENCE   2  (bases 1 to 4763)
  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"
COMMENT     Sequence Label: pCI_FLAG-GFP(Y39TAG)-6His
FEATURES             Location/Qualifiers
     source          1..4763
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(435..1023)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(515..534)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1194..1985,1986..2054))
                     /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     1817..1836
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2055..2159)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2227..2245
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2283..2305)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2405..2424
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2491..2946)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2628..2649)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2840..2859
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3127..3248
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3164..3183)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3218..3237
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(3291..3308)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(3309..4019,4020..4022))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT*GKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3351..3372)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3700..3719
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3959..3980
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(4023..4046)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(4073..4091)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4134..4153)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          4136..4268
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     complement(4383..4407)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(4404..4607)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4433..4453)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        join(4608..4763,1..224)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac
       61 tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg
      121 cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga
      181 ccccgtaatt gattactatt aataactagt caataatcaa tgccaacatg gcggtcatat
      241 tggacatgag ccaatataaa tgtacatatt atgatataga tacaacgtat gcaatggcca
      301 atagccaata ttgatttatg ctatataacc aatgaataat atggctaatg gccaatattg
      361 aagatctgtc gagccatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
      421 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
      481 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
      541 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
      601 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
      661 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
      721 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
      781 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
      841 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
      901 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
      961 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1021 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1081 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     1141 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     1201 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1261 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1321 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1381 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     1441 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1501 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1561 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     1621 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1681 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1741 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1801 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     1861 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1921 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1981 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2041 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     2101 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     2161 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     2221 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     2281 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     2341 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     2401 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     2461 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat
     2521 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     2581 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     2641 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     2701 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     2761 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     2821 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     2881 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     2941 gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     3001 ttcgctatta cgccagcccg gatccttatc gattttacca catttgtaga ggttttactt
     3061 gctttaaaaa acctcccaca tctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     3121 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3181 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3241 gtatcttatc atgtctgctc gaagcggccg cccgggtcga ctctagatca gtgatggtga
     3301 tggtgatgct tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
     3361 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc
     3421 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
     3481 tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttggccttg
     3541 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
     3601 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     3661 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     3721 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     3781 tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc
     3841 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc
     3901 ttgccctagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     3961 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     4021 actttatcat catcatcttt gtaatccatc gcggcggaat tctcgaggct agcctatagt
     4081 gagtcgtatt aagtactcta gccttaagag ctgtaattga actgggagtg gacacctgtg
     4141 gagagaaagg caaagtggat gtcagtaaga ccaataggtg cctatcagaa acgcaagagt
     4201 cttctctgtc tcgacaagcc cagtttctat tggtctcctt aaacctgtct tgtaaccttg
     4261 atacttacct gcccagtgcc tcacgaccaa cttctgcagc ttaagttcga gactgttgtg
     4321 tcagaagcac tgactgcgtt agcaatttaa ctgtgataaa ctaccgcaat aaagcttcta
     4381 gtgatctgac ggttcactaa acgagctctg cttatataga cctcccaccg tacacgccta
     4441 ccgcccattt gcgtcaacgg ggcggggtta ttacgacatt ttggaaagtc ccgttgattt
     4501 tggtgccaaa acaaactccc attgacgtca atggggtgga gacttggaaa tccccgtgag
     4561 tcaaaccgct atccacgccc attggtgtac tgccaaaacc gcatcaccat ggtaatagcg
     4621 atgactaata cgtagatgta ctgccaagta ggaaagtccc gtaaggtcat gtactgggca
     4681 taatgccagg cgggccattt accgtcattg acgtcaatag ggggcggact tggcatatga
     4741 tacacttgat gtactgccaa gtg
//
