LOCUS       sequence-214108-        6646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses zinc alpha-2 glycoprotein (ZAG) and dTomato (dT) reporter 
            where both sequences are separated by a T2A self-cleaving peptide 
            sequence as a result, the dT is not fused with the ZAG protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6646)
  AUTHORS   Elattar S, Dimri M, Satyanarayana A
  TITLE     The tumor secretory factor ZAG promotes white adipose tissue 
            browning and energy wasting.
  JOURNAL   FASEB J. 2018 Mar 23:fj201701465RR. doi: 10.1096/fj.201701465RR.
  PUBMED    29570397
REFERENCE   2  (bases 1 to 6646)
  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: "FASEB J. 
            2018 Mar 23:fj201701465RR. doi: 10.1096/fj.201701465RR."
COMMENT     Sequence Label: pRP(Exp)-CMV> ORF_924bp
            (Azgp1):T2A:dTomato:IR...
FEATURES             Location/Qualifiers
     source          1..6646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..49
                     /note="synthetic polyadenylation signal"
     misc_feature    63..154
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     103..122
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     protein_bind    190..210
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        271..574
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        575..778
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     729..749
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    799..823
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      824..833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1751..1804
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1805..2506
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(1938..1956)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2206..2225
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2400..2423
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(2510..2534)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(2535..3109,3110..3112,3113..3121)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2702..2719)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2929..2948
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3122..3721
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3122..3141)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3618..3638
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(3722..3742)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3770..3891
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3781..3800)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3835..3854
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4139..4156)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4310..4898)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4390..4409)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5069..5860,5861..5929))
                     /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     5692..5711
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5930..6034)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6061..6516
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6148..6167)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6358..6379
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta
       61 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc
      121 cccagtgcaa gtgcaggtgc cagaacattt ctctatcgat aggtaccgag ctcttacgcg
      181 tgctagcatc aactttgtat agaaaagttg tagttattaa tagtaatcaa ttacggggtc
      241 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      301 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      361 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      421 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      481 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      541 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      601 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      661 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      721 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
      781 tttagtgaac cgtcagatcc aagtttgtac aaaaaagcag gctgccacca tggtgcctgt
      841 cctgctgtcc ctgcctctcc ttctgggtcc tgcagtcttt caggagactg ggtcttatta
      901 tctgaccttt ctctacaccg ggttgtccag acccagcaaa ggttttccga ggtttcaagc
      961 cactgcattt ctcaatgacc aggccttctt ccactacaac agcaacagcg ggaaggcaga
     1021 gcctgtggga ccttggagcc aggtggaagg aatggaggac tgggagaagg aaagccagct
     1081 tcagagggcc agggaggaga tcttccttgt gaccctgaaa gacatcatgg actattacaa
     1141 ggacactaca gggtctcaca cctttcaggg aatgtttggt tgcgagatca caaataacag
     1201 aagtagtgga gctgtctgga ggtatgccta cgacggagag gatttcatcg aattcaacaa
     1261 agaaatccca gcttggatcc ccttagaccc agcagctgca aacaccaagc taaagtggga
     1321 agcagaaaag gtctacgtgc agcgagccaa ggcataccta gaggaggagt gtcctgaaat
     1381 gctgaagagg tacctgaact acagtcgatc tcacctggac cgaatagatc ctcccactgt
     1441 gacaatcacc agccgtgtga tcccaggagg aaacagaata ttcaaatgcc tggcctatgg
     1501 cttctaccca caaagaatta gtctgcactg gaacaaggcc aacaagaagc tagcatttga
     1561 accagaaaga ggtgtttttc ccaatggaaa tggcacttac ctctcctggg cagaggtgga
     1621 agtctcccca caggacatag accccttctt ctgcctcata gatcacaggg ggttttccca
     1681 atctctctcg gtgcagtggg ataggacaag aaaagtaaag gatgaaaaca atgttgtagc
     1741 tcagcctcag gagggcaggg gaagtcttct aacatgcggg gacgtggagg aaaatcccgg
     1801 ccccatggtg agcaagggcg aggaggtcat caaagagttc atgcgcttca aggtgcgcat
     1861 ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     1921 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg
     1981 ggacatcctg tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga
     2041 catccccgat tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     2101 cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct
     2161 gatctacaag gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa
     2221 gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa
     2281 gggcgagatc caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa
     2341 gaccatctac atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa
     2401 gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga
     2461 gggccgccac cacctgttcc tgtacggcat ggacgagctg tacaagtaaa cccagctttc
     2521 ttgtacaaag tgggcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg
     2581 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc
     2641 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc
     2701 cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa
     2761 gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct
     2821 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca
     2881 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca
     2941 agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat
     3001 ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc
     3061 cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac
     3121 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     3181 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     3241 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     3301 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     3361 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     3421 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     3481 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     3541 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     3601 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     3661 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     3721 acaactttat tatacatagt tgatggccgg ccgcttcgag cagacatgat aagatacatt
     3781 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     3841 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     3901 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     3961 taaaacctct acaaatgtgg taaaatcgat aaggatccgt cgaccgatgc ccttgagagc
     4021 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
     4081 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct tccgcttcct
     4141 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4201 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4261 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4321 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4381 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4441 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4501 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4561 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4621 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4681 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4741 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4801 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4861 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4921 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4981 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5041 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5101 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5161 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5221 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5281 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5341 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5401 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5461 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5521 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5581 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5641 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5701 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5761 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5821 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5881 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5941 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6001 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6061 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     6121 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     6181 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     6241 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     6301 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     6361 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     6421 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     6481 acgcgaattt taacaaaata ttaacgctta caatttgcca ttcgccattc aggctgcgca
     6541 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagccc aagctaccat
     6601 gataagtaag taatattaag gtacgggagg tacttggagc ggccgc
//
