LOCUS       sequence-349771-        8293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding ITGB2 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8293)
  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 8293)
  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: ITGB2_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..8293
                     /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(3015..3039)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3065..3106
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3146..3165)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3190..3238
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3440..3868
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3527..3546)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3737..3758
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3883..4382
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3918..3941)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4315..4334
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        4396..4443
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(4462..4464,4465..4467,4468..5178)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4507..4528)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4768..4787)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5115..5136
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5179..5574
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5732..5853
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5769..5788)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5823..5842
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5902..5918)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5902..5918)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5915..5937)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5926..5942
                     /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(5950..5956,5957..5974,5975..5980))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5995..6016
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6133..6150)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6304..6892)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6384..6403)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7063..7854,7855..7923))
                     /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     7686..7705
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7924..8028)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8103..8122)
                     /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 accatgctgg gcctgcgccc
      721 cccacttctc gccctggtgg ggctgctctc cctcgggtgc gtcctctctc aggagtgcac
      781 gaagttcaag gtcagcagct gccgggaatg catcgagtcg gggcccggct gcacctggtg
      841 ccagaagctg aacttcacag ggccggggga tcctgactcc attcgctgcg acacccggcc
      901 acagctgctc atgaggggct gtgcggctga cgacatcatg gaccccacaa gcctcgctga
      961 aacccaggaa gaccacaatg ggggccagaa gcagctgtcc ccacaaaaag tgacgcttta
     1021 cctgcgacca ggccaggcag cagcgttcaa cgtgaccttc cggcgggcca agggctaccc
     1081 catcgacctg tactatctga tggacctctc ctactccatg cttgatgacc tcaggaatgt
     1141 caagaagcta ggtggcgacc tgctccgggc cctcaacgag atcaccgagt ccggccgcat
     1201 tggcttcggg tccttcgtgg acaagaccgt gctgccgttc gtgaacacgc accctgataa
     1261 gctgcgaaac ccatgcccca acaaggagaa agagtgccag cccccgtttg ccttcaggca
     1321 cgtgctgaag ctgaccaaca actccaacca gtttcagacc gaggtcggga agcagctgat
     1381 ttccggaaac ctggatgcac ccgagggtgg gctggacgcc atgatgcagg tcgccgcctg
     1441 cccggaggaa atcggctggc gcaacgtcac gcggctgctg gtgtttgcca ctgatgacgg
     1501 cttccatttc gcgggcgacg ggaagctggg cgccatcctg acccccaacg acggccgctg
     1561 tcacctggag gacaacttgt acaagaggag caacgaattc gactacccat cggtgggcca
     1621 gctggcgcac aagctggctg aaaacaacat ccagcccatc ttcgcggtga ccagtaggat
     1681 ggtgaagacc tacgagaaac tcaccgagat catccccaag tcagccgtgg gggagctgtc
     1741 tgaggactcc agcaatgtgg tccatctcat taagaatgct tacaataaac tctcctccag
     1801 ggtattcctg gatcacaacg ccctccccga caccctgaaa gtcacctacg actccttctg
     1861 cagcaatgga gtgacgcaca ggaaccagcc cagaggtgac tgtgatggcg tgcagatcaa
     1921 tgtcccgatc accttccagg tgaaggtcac ggccacagag tgcatccagg agcagtcgtt
     1981 tgtcatccgg gcgctgggct tcacggacat agtgaccgtg caggtccttc cccagtgtga
     2041 gtgccggtgc cgggaccaga gcagagaccg cagcctctgc catggcaagg gcttcttgga
     2101 gtgcggcatc tgcaggtgtg acactggcta cattgggaaa aactgtgagt gccagacaca
     2161 gggccggagc agccaggagc tggaaggaag ctgccggaag gacaacaact ccatcatctg
     2221 ctcagggctg ggggactgtg tctgcgggca gtgcctgtgc cacaccagcg acgtccccgg
     2281 caagctgata tacgggcagt actgcgagtg tgacaccatc aactgtgagc gctacaacgg
     2341 ccaggtctgc ggcggcccgg ggagggggct ctgcttctgc gggaagtgcc gctgccaccc
     2401 gggctttgag ggctcagcgt gccagtgcga gaggaccact gagggctgcc tgaacccgcg
     2461 gcgtgttgag tgtagtggtc gtggccggtg ccgctgcaac gtatgcgagt gccattcagg
     2521 ctaccagctg cctctgtgcc aggagtgccc cggctgcccc tcaccctgtg gcaagtacat
     2581 ctcctgcgcc gagtgcctga agttcgaaaa gggccccttt gggaagaact gcagcgcggc
     2641 gtgtccgggc ctgcagctgt cgaacaaccc cgtgaagggc aggacctgca aggagaggga
     2701 ctcagagggc tgctgggtgg cctacacgct ggagcagcag gacgggatgg accgctacct
     2761 catctatgtg gatgagagcc gagagtgtgt ggcaggcccc aacatcgccg ccatcgtcgg
     2821 gggcaccgtg gcaggcatcg tgctgatcgg cattctcctg ctggtcatct ggaaggctct
     2881 gatccacctg agcgacctcc gggagtacag gcgctttgag aaggagaagc tcaagtccca
     2941 gtggaacaat gataatcccc ttttcaagag cgccaccacg acggtcatga accccaagtt
     3001 tgctgagagt tagaacccag ctttcttgta caaagtggtt gatctagagg gcccgcggtt
     3061 cgaaggtaag cctatcccta accctctcct cggtctcgat tctacgcgta ccggttagta
     3121 atgagtttaa acgggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     3181 gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc gtttgttcat
     3241 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     3301 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     3361 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcagatct gcgcagctgg
     3421 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3481 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3541 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3601 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3661 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3721 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3781 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3841 ctgatttaac aaaaatttaa cgcgaattat cctcgagcta ccgggtaggg gaggcgcttt
     3901 tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa
     3961 gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct
     4021 ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc
     4081 cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc
     4141 agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc
     4201 agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc
     4261 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
     4321 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt
     4381 cgactctaga cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
     4441 gacaaggtga ggaactaaac catggtgagc aagggcgagg agctgttcac cggggtggtg
     4501 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     4561 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     4621 ctgcccgtgc cctggcccac cctcgtgacc accttcacct acggcgtgca gtgcttcgcc
     4681 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     4741 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     4801 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     4861 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa ggtctatatc
     4921 accgccgaca agcagaagaa cggcatcaag gtgaacttca agacccgcca caacatcgag
     4981 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     5041 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     5101 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     5161 atggacgagc tgtacaaggc caagcctttg tctcaagaag aatccaccct cattgaaaga
     5221 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct
     5281 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct
     5341 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt
     5401 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag
     5461 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg
     5521 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaagcactt
     5581 cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg ccgccttcta
     5641 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     5701 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     5761 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     5821 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     5881 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5941 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     6001 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6061 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6121 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6181 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6241 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6301 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6361 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6421 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6481 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6541 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6601 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6661 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6721 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6781 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6841 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6901 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6961 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7021 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     7081 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7141 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7201 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     7261 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7321 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     7381 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagcttcg gttcccaacg
     7441 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7501 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7561 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7621 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7681 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7741 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7801 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7861 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7921 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7981 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     8041 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     8101 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     8161 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     8221 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     8281 agatatacgc gtt
//
