LOCUS       sequence-349695-        7033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding CRYZL1 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7033)
  AUTHORS   Moyer AJ, Fernandez FX, Li Y, Klinedinst DK, Florea LD, Kazuki Y, 
            Oshimura M, Reeves RH
  TITLE     Overexpression screen of chromosome 21 genes reveals modulators of 
            Sonic hedgehog signaling relevant to Down syndrome.
  JOURNAL   Dis Model Mech. 2023 Apr 1;16(4):dmm049712. doi: 10.1242/dmm.049712.
            Epub 2023 Apr 13.
  PUBMED    36995257
REFERENCE   2  (bases 1 to 7033)
  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: "Dis Model 
            Mech."; date: "2023-04-1"; volume: "16(4):dmm049712. doi"; pages: " 
            10.1242/dmm.049712. Epub 2023 Apr 13"
COMMENT     Sequence Label: CRYZL1_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..7033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(180..199)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        371..750
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        751..954
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     905..925
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     999..1018
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        999..1017
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1047..1071
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2125..2149)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2175..2216
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2256..2275)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2300..2348
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2550..2978
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2637..2656)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2847..2868
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2993..3492
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3028..3051)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3425..3444
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3506..3553
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3572..3574,3575..3577,3578..4288)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3617..3638)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3878..3897)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4225..4246
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4289..4684
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4842..4963
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4879..4898)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4933..4952
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5012..5028)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5012..5028)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5025..5047)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5036..5052
                     /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(5060..5066,5067..5084,5085..5090))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5105..5126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5243..5260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5414..6002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5494..5513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6173..6964,6965..7033))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPKLNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6796..6815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
      181 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
      241 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
      301 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
      361 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      421 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      481 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      541 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      601 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      661 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      721 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      781 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      841 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      901 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
      961 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag
     1021 acccaagctg gctagttaag ctatcaacaa gtttgtacaa aaaagcaggc accatgaaag
     1081 gcttatattt ccaacagagt tccacagatg aagaaataac atttgtattt caagaaaagg
     1141 aagatcttcc tgttacagag gataactttg tgaaacttca agttaaagct tgtgctctga
     1201 gccagataaa tacaaagctt ctggcagaaa tgaagatgaa aaaggattta tttcctgttg
     1261 ggagagaaat tgctggaatt gtattagatg ttggaagcaa ggtatcattc tttcaaccag
     1321 atgatgaagt agttggaatt ttgcccctgg actctgaaga ccctggactt tgtgaagttg
     1381 ttagagtaca tgagcattac ttggttcata aaccagaaaa ggtcacatgg acggaagcag
     1441 caggaagcat tcgggatgga gtgcgtgcct atacagctct gcattatctt tctcatctct
     1501 ctcctggaaa atcagtgctg ataatggatg gagcaagtgc atttggtaca atagctattc
     1561 agttagcaca tcatagagga gccaaagtga tttcaacagc atgcagcctt gaagataagc
     1621 agtgccttga aagattcaga cctcccatag cccgagtgat tgatgtatct aatgggaaag
     1681 ttcatgttgc tgaaagctgt ttggaagaaa caggtggcct gggagtagat attgtcctag
     1741 atgctggagt gagattatat agtaaagatg atgaaccagc tgtaaaacta caactactac
     1801 cacataaaca tgatatcatc acacttcttg gtgttggagg ccactgggta acaacagaag
     1861 aaaaccttca gttggatcct ccagatagcc actgcctttt cctcaaggga gcaacgttag
     1921 ctttcctgaa tgatgaagtt tggaatttgt caaatgtaca acagggaaaa tatctttgta
     1981 tcttaaagga tgtgatggag aagttatcaa ctggtgtttt cagacctcag ttggatgaac
     2041 ccattccact gtatgaggca aaagtttcca tggaagctgt tcagaaaaat caaggaagaa
     2101 aaaagcaagt tgttcaattt tagaacccag ctttcttgta caaagtggtt gatctagagg
     2161 gcccgcggtt cgaaggtaag cctatcccta accctctcct cggtctcgat tctacgcgta
     2221 ccggttagta atgagtttaa acgggggagg ctaactgaaa cacggaagga gacaataccg
     2281 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc
     2341 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     2401 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     2461 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcagatct
     2521 gcgcagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2581 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2641 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2701 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2761 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2821 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2881 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2941 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattat cctcgagcta ccgggtaggg
     3001 gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg
     3061 cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg
     3121 gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc
     3181 ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact
     3241 agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc
     3301 ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc
     3361 gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc
     3421 ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct
     3481 ccgggccttt cgactctaga cacgtgttga caattaatca tcggcatagt atatcggcat
     3541 agtataatac gacaaggtga ggaactaaac catggtgagc aagggcgagg agctgttcac
     3601 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     3661 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     3721 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcacct acggcgtgca
     3781 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     3841 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     3901 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     3961 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     4021 ggtctatatc accgccgaca agcagaagaa cggcatcaag gtgaacttca agacccgcca
     4081 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     4141 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     4201 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     4261 cactctcggc atggacgagc tgtacaaggc caagcctttg tctcaagaag aatccaccct
     4321 cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc
     4381 cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac
     4441 tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa
     4501 cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg
     4561 gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga
     4621 tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg
     4681 ctaagcactt cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg
     4741 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4801 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4861 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4921 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4981 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5041 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5101 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5161 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5221 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5281 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5341 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5401 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5461 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5521 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5581 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5641 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5701 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5761 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5821 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5881 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5941 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6001 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6061 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6121 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6181 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6241 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6301 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6361 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6421 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6481 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagcttcg
     6541 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6601 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6661 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6721 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6781 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6841 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6901 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6961 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7021 gttgaatact cat
//
