LOCUS       sequence-213403-        6817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of turboGFP-CRYZ with FLAG tag, Flp-In T-Rex 
            system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6817)
  TITLE     Mammalian expression plasmids based on pcDNA5/FRT/TO family
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6817)
  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            "Unpublished"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: pcDNA5/FRT/TO/Flag-tGFP-Cryz
FEATURES             Location/Qualifiers
     source          1..6817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      749..758
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             758..781
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             788..1483
                     /codon_start=1
                     /product="improved green fluorescent protein from 
                     Pontellina plumata (Evdokimov et al., 2006)"
                     /label=TurboGFP
                     /note="mammalian codon-optimized"
                     /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKM
                     KSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVL
                     HVSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNDLDGSFTR
                     TFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAFRRVEEDHSNTELGIVEYQHAF
                     KTPDADAGEE"
     primer_bind     complement(2535..2552)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2541..2765
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2898..2917)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3049..3096
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3104..4126
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3104..4126
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4256..4377
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4293..4312)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4347..4366
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4426..4442)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4426..4442)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4439..4461)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4450..4466
                     /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(4474..4480,4481..4498,4499..4504))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4519..4540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4657..4674)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4828..5416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4908..4927)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5587..6378,6379..6447))
                     /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     6210..6229
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6448..6552)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6627..6646)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagctgc caccatggac tacaaggacg acgatgacaa
      781 gaagcttatg gagagcgacg agagcggcct gcccgccatg gagatcgagt gccgcatcac
      841 cggcaccctg aacggcgtgg agttcgagct ggtgggcggc ggagagggca cccccgagca
      901 gggccgcatg accaacaaga tgaagagcac caaaggcgcc ctgaccttca gcccctacct
      961 gctgagccac gtgatgggct acggcttcta ccacttcggc acctacccca gcggctacga
     1021 gaaccccttc ctgcacgcca tcaacaacgg cggctacacc aacacccgca tcgagaagta
     1081 cgaggacggc ggcgtgctgc acgtgagctt cagctaccgc tacgaggccg gccgcgtgat
     1141 cggcgacttc aaggtgatgg gcaccggctt ccccgaggac agcgtgatct tcaccgacaa
     1201 gatcatccgc agcaacgcca ccgtggagca cctgcacccc atgggcgata acgatctgga
     1261 tggcagcttc acccgcacct tcagcctgcg cgacggcggc tactacagct ccgtggtgga
     1321 cagccacatg cacttcaaga gcgccatcca ccccagcatc ctgcagaacg ggggccccat
     1381 gttcgccttc cgccgcgtgg aggaggatca cagcaacacc gagctgggca tcgtggagta
     1441 ccagcacgcc ttcaagaccc cggatgcaga tgccggtgaa gaaagagttg gtaccgagct
     1501 cggatccatg gcgactggac agaagttgat gagagctgtt agagtttttg aatttggtgg
     1561 gccagaagtc ctgaaattgc gatcagatat tgcagtaccg attccaaaag accatcaggt
     1621 tctaatcaag gtccatgcat gtggtgtcaa ccccgtggag acatacattc gctctggtac
     1681 ttatagtaga aaaccactct taccctatac tcctggctca gatgtggctg gggtgataga
     1741 agctgttgga gataatgcat ctgctttcaa gaaaggtgac agagttttca ctagcagcac
     1801 gatctctggg ggttatgcag agtatgctct tgcagcagac cacactgttt acaaactacc
     1861 tgaaaaactg gactttaaac aaggagctgc catcggcatt ccatatttta ctgcttatcg
     1921 agctctgatc cacagtgcct gtgtgaaagc tggagagagt gttctggttc atggggcaag
     1981 tggaggagtt ggattagcag catgccaaat tgctagagct tatggcttaa agattttggg
     2041 cactgctggt actgaggaag gacaaaagat tgttttgcaa aatggagccc atgaagtgtt
     2101 caatcacaga gaagtgaatt acattgataa aattaagaag tatgttggtg agaaaggaat
     2161 tgatataatt attgaaatgt tagctaatgt aaatcttagt aaagacttga gtcttctgtc
     2221 acatggagga cgagtgatag ttgttggcag cagaggtact attgaaataa acccacgaga
     2281 caccatggca aaggagtcga gtataattgg agttactctc ttttcctcaa ccaaggagga
     2341 atttcagcaa tatgcagcag cccttcaagc tggaatggaa attggctggt tgaaacctgt
     2401 gataggttct caatatccat tggagaaggt ggccgaggct catgaaaata tcattcatgg
     2461 tagtggggct actggaaaaa tgattcttct cttatgactc gagtctagag ggcccgttta
     2521 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2581 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2641 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2701 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2761 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     2821 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2881 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2941 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3001 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt
     3061 ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac
     3121 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     3181 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     3241 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     3301 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct
     3361 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     3421 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     3481 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     3541 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     3601 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     3661 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     3721 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     3781 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     3841 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     3901 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     3961 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     4021 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     4081 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga
     4141 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4201 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4261 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4321 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4381 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4441 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4501 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4561 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4621 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4681 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4741 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4801 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4861 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4921 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4981 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5041 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5101 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5161 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5221 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5281 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5341 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5401 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5461 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5521 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5581 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5641 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5701 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5761 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5821 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5881 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5941 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6001 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6061 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6121 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6181 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6241 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6301 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6361 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6421 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6481 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6541 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     6601 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6661 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     6721 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
     6781 gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
