LOCUS       sequence-207758-        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  JOURNAL   Unpublished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            "Unpublished"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"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      983..992
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             992..1015
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1022..1717
                     /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(2769..2786)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2775..2999
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3132..3151)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3283..3330
                     /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             3338..4360
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3338..4360
                     /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    4490..4611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4527..4546)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4581..4600
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4660..4676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4660..4676)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4673..4695)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4684..4700
                     /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(4708..4714,4715..4732,4733..4738))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4753..4774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4891..4908)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5062..5650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5142..5161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5821..6612,6613..6681))
                     /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     6444..6463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6682..6786)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag ctgccaccat ggactacaag gacgacgatg acaagaagct
     1021 tatggagagc gacgagagcg gcctgcccgc catggagatc gagtgccgca tcaccggcac
     1081 cctgaacggc gtggagttcg agctggtggg cggcggagag ggcacccccg agcagggccg
     1141 catgaccaac aagatgaaga gcaccaaagg cgccctgacc ttcagcccct acctgctgag
     1201 ccacgtgatg ggctacggct tctaccactt cggcacctac cccagcggct acgagaaccc
     1261 cttcctgcac gccatcaaca acggcggcta caccaacacc cgcatcgaga agtacgagga
     1321 cggcggcgtg ctgcacgtga gcttcagcta ccgctacgag gccggccgcg tgatcggcga
     1381 cttcaaggtg atgggcaccg gcttccccga ggacagcgtg atcttcaccg acaagatcat
     1441 ccgcagcaac gccaccgtgg agcacctgca ccccatgggc gataacgatc tggatggcag
     1501 cttcacccgc accttcagcc tgcgcgacgg cggctactac agctccgtgg tggacagcca
     1561 catgcacttc aagagcgcca tccaccccag catcctgcag aacgggggcc ccatgttcgc
     1621 cttccgccgc gtggaggagg atcacagcaa caccgagctg ggcatcgtgg agtaccagca
     1681 cgccttcaag accccggatg cagatgccgg tgaagaaaga gttggtaccg agctcggatc
     1741 catggcgact ggacagaagt tgatgagagc tgttagagtt tttgaatttg gtgggccaga
     1801 agtcctgaaa ttgcgatcag atattgcagt accgattcca aaagaccatc aggttctaat
     1861 caaggtccat gcatgtggtg tcaaccccgt ggagacatac attcgctctg gtacttatag
     1921 tagaaaacca ctcttaccct atactcctgg ctcagatgtg gctggggtga tagaagctgt
     1981 tggagataat gcatctgctt tcaagaaagg tgacagagtt ttcactagca gcacgatctc
     2041 tgggggttat gcagagtatg ctcttgcagc agaccacact gtttacaaac tacctgaaaa
     2101 actggacttt aaacaaggag ctgccatcgg cattccatat tttactgctt atcgagctct
     2161 gatccacagt gcctgtgtga aagctggaga gagtgttctg gttcatgggg caagtggagg
     2221 agttggatta gcagcatgcc aaattgctag agcttatggc ttaaagattt tgggcactgc
     2281 tggtactgag gaaggacaaa agattgtttt gcaaaatgga gcccatgaag tgttcaatca
     2341 cagagaagtg aattacattg ataaaattaa gaagtatgtt ggtgagaaag gaattgatat
     2401 aattattgaa atgttagcta atgtaaatct tagtaaagac ttgagtcttc tgtcacatgg
     2461 aggacgagtg atagttgttg gcagcagagg tactattgaa ataaacccac gagacaccat
     2521 ggcaaaggag tcgagtataa ttggagttac tctcttttcc tcaaccaagg aggaatttca
     2581 gcaatatgca gcagcccttc aagctggaat ggaaattggc tggttgaaac ctgtgatagg
     2641 ttctcaatat ccattggaga aggtggccga ggctcatgaa aatatcattc atggtagtgg
     2701 ggctactgga aaaatgattc ttctcttatg actcgagtct agagggcccg tttaaacccg
     2761 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2821 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2881 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2941 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3001 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3061 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3121 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3181 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3241 cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag
     3301 ttcctattct ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac
     3361 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
     3421 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     3481 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     3541 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta
     3601 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     3661 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     3721 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     3781 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     3841 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     3901 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     3961 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     4021 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     4081 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     4141 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     4201 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     4261 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     4321 aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga
     4381 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4441 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4501 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4561 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4621 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4681 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4741 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4801 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4861 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4921 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4981 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5041 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5101 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5161 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5221 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5281 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5341 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5401 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5461 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5521 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5581 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5641 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5701 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5761 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5821 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5881 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5941 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6001 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6061 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6121 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6181 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6241 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6301 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6361 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6421 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6481 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6541 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6601 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6661 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6721 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6781 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
