LOCUS       sequence-349705-        7453 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression vector encoding SLC19A1 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7453)
  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 7453)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: SLC19A1_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..7453
                     /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"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    677..701
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2175..2199)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2225..2266
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2306..2325)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2350..2398
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2600..3028
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2687..2706)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2897..2918
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3043..3542
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3078..3101)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3475..3494
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3556..3603
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3622..3624,3625..3627,3628..4338)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3667..3688)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3928..3947)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4275..4296
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4339..4734
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4892..5013
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4929..4948)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4983..5002
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5062..5078)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5062..5078)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5075..5097)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5086..5102
                     /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(5110..5116,5117..5134,5135..5140))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5155..5176
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5293..5310)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5464..6052)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5544..5563)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6223..7014,7015..7083))
                     /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     6846..6865
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7084..7188)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7263..7282)
                     /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 agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagttaag ctatcaacaa gtttgtacaa aaaagcaggc accatggtgc cctccagccc
      721 agcggtggag aagcaggtgc ccgtggaacc tgggcctgac cccgagctcc ggtcctggcg
      781 gcgcctcgtg tgctaccttt gcttctacgg cttcatggcg cagatacggc caggggagag
      841 cttcatcacc ccctacctcc tggggcccga caagaacttc acgcgggagc aggtcacgaa
      901 cgagatcacg ccggtgctgt cgtactccta cctggccgtg ctggtgcccg tgttcctgct
      961 caccgactac ctgcgctaca cgccggtgct gctgctgcag gggctcagct tcgtgtcggt
     1021 gtggctgctg ctgctgctgg gccactcggt ggcgcacatg cagctcatgg agctcttcta
     1081 cagcgtcacc atggccgcgc gcatcgccta ttcctcctac atcttctctc tcgtgcggcc
     1141 cgcgcgctac cagcgtgtgg ccggctactc gcgcgctgcg gtgctgctgg gcgtgttcac
     1201 cagctccgtg ctgggccagc tgctggtcac tgtgggccga gtctccttct ccacgctcaa
     1261 ctacatctcg ctggccttcc tcaccttcag cgtggtcctc gccctcttcc tgaagcgccc
     1321 caagcgcagc ctcttcttca accgcgacga ccgggggcgg tgcgaaacct cggcttcgga
     1381 gctggagcgc atgaatcccg gcccaggcgg gaagctggga cacgccctgc gggtggcctg
     1441 tggggactca gtgctggcgc ggatgctgcg ggagctgggg gacagcctgc ggcggccgca
     1501 gctgcgcctg tggtccctct ggtgggtctt caactcggcc ggctactacc tggtggtcta
     1561 ctacgtgcac atcctgtgga acgaggtgga ccccaccacc aacagtgcgc gggtctacaa
     1621 cggcgcggca gatgctgcct ccacgctgct gggcgccatc acgtccttcg ccgcgggctt
     1681 cgtgaagatc cgctgggcgc gctggtccaa gctgctcatc gcgggcgtca cggccacgca
     1741 ggcggggctg gtcttccttc tggcgcacac gcgccacccg agcagcatct ggctgtgcta
     1801 tgcggccttc gtgctgttcc gcggctccta ccagttcctc gtgcccatcg ccacctttca
     1861 gattgcatct tctctgtcta aagagctctg tgccctggtc ttcggggtca acacgttctt
     1921 tgccaccatc gtcaagacca tcatcacttt cattgtctcg gacgtgcggg gcctgggcct
     1981 cccggtccgc aagcagaatg aagaactgca tgtggcttct ctgtctctgt ggaaaagcca
     2041 tctcaggttg gcggcagaca cattgtcatc agaggggagc agcggctctg gtcctcggag
     2101 ctggttcctc tctcccaccc taagggcagc cctccatggt cctgtctgtc cttctgaagt
     2161 gtgtccatcc tagaacccag ctttcttgta caaagtggtt gatctagagg gcccgcggtt
     2221 cgaaggtaag cctatcccta accctctcct cggtctcgat tctacgcgta ccggttagta
     2281 atgagtttaa acgggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     2341 gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc gtttgttcat
     2401 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     2461 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     2521 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcagatct gcgcagctgg
     2581 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2641 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2701 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2761 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2821 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2881 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2941 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3001 ctgatttaac aaaaatttaa cgcgaattat cctcgagcta ccgggtaggg gaggcgcttt
     3061 tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa
     3121 gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct
     3181 ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc
     3241 cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc
     3301 agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc
     3361 agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc
     3421 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
     3481 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt
     3541 cgactctaga cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
     3601 gacaaggtga ggaactaaac catggtgagc aagggcgagg agctgttcac cggggtggtg
     3661 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     3721 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     3781 ctgcccgtgc cctggcccac cctcgtgacc accttcacct acggcgtgca gtgcttcgcc
     3841 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     3901 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     3961 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     4021 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa ggtctatatc
     4081 accgccgaca agcagaagaa cggcatcaag gtgaacttca agacccgcca caacatcgag
     4141 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     4201 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     4261 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     4321 atggacgagc tgtacaaggc caagcctttg tctcaagaag aatccaccct cattgaaaga
     4381 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct
     4441 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct
     4501 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt
     4561 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag
     4621 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg
     4681 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaagcactt
     4741 cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg ccgccttcta
     4801 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4861 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4921 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4981 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     5041 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5101 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5161 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5221 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5281 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5341 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5401 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5461 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5521 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5581 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5641 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5701 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5761 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5821 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5881 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5941 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6001 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6061 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6121 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6181 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6241 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6301 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6361 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6421 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6481 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6541 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagcttcg gttcccaacg
     6601 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6661 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6721 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6781 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6841 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6901 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6961 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7021 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7081 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7141 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7201 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     7261 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     7321 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     7381 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     7441 agatatacgc gtt
//
