LOCUS       sequence-249150-        7417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing human Rad9b frameshift mutation found in a neural tube 
            defect patient.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7417)
  AUTHORS   Cao X, Tian T, Steele JW, Cabrera RM, Aguiar-Pulido V, Wadhwa S, 
            Bhavani N, Bi P, Gargurevich NH, Hoffman EN, Cai CQ, Marini NJ, Yang
            W, Shaw GM, Ross ME, Finnell RH, Lei Y
  TITLE     Loss of RAD9B impairs early neural development and contributes to 
            the risk for human spina bifida.
  JOURNAL   Hum Mutat. 2020 Apr;41(4):786-799. doi: 10.1002/humu.23969. Epub 
            2020 Jan 16.
  PUBMED    31898828
REFERENCE   2  (bases 1 to 7417)
  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"; doi: 
            "10.1002/humu.23969"; journalName: "Hum Mutat"; date: "2020-04"; 
            volume: "41"; issue: "4"; pages: "786-799"
COMMENT     Sequence Label: R9-FS1
FEATURES             Location/Qualifiers
     source          1..7417
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        142..521
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        522..725
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     676..696
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     770..789
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        770..788
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             830..1543
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(830..832,833..1543)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(872..893)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1133..1152)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1480..1501
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2915..2932)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2921..3145
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3191..3619
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3278..3297)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3488..3509
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3628..3648)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3633..3962
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3813..3948
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3875..3894
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4029..4823
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4083..4102)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4693..4712
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4997..5118
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5034..5053)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5088..5107
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5167..5183)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5167..5183)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5180..5202)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5191..5207
                     /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(5215..5221,5222..5239,5240..5245))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5260..5281
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5398..5415)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5569..6157)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5649..5668)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6328..7119,7120..7188))
                     /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     6951..6970
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7189..7293)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7368..7387)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
       61 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
      121 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      181 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      241 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      301 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      361 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      421 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      481 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      541 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      601 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      661 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      721 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
      781 actataggga gacccaagct ggctagcgtt taaacttaag cttgccacca tgagcaaggg
      841 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      901 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
      961 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1021 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1081 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1141 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1201 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1261 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1321 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1381 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     1441 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1501 gaccgccgcc gggatcactc acggcatgga cgagctgtac aagggtacca tggcagccat
     1561 gctgaagtgc gtgatgagcg gcagtcaggt gaaagtattt gggaaagcag ttcaagctct
     1621 atcacgaatt agtgacgagt tctggctaga cccatctaaa aaaggtcttg ctctaagatg
     1681 tgtgaattct tctcggtcag catatggatg tgtcctgttc tctcctgtgt tttttcagca
     1741 ttatcaatgg tcagctttag tgaaaatgag tgaaaatgaa cttgacacaa cactgcattt
     1801 aaaatgcaaa ttgggaatga agtcaatttt gcccatcttt agatgtctga attcccttga
     1861 aagaaatata gagaagtgca gaatattcac cagatctgat aaatgcaaag tagttattca
     1921 attcttctac agacatggta ttaaaagaac tcataatata tgttttcaag aaagtcagcc
     1981 tttgcaagtt atttttgaca agaatgtttg tactaatacg ctaatgattc aaccaagatt
     2041 gcttgctgat gccattgttc tttttacatc aagtcaagag gaagttactc ttgctgttac
     2101 tccactgaat ttttgcctca agagttctaa tgaggaatca atggatttga gcaatgctgt
     2161 acacagtgag atgtttgttg gctcagatga gtttgacttc tttcaaattg gaatggacac
     2221 tgagataaca ttttgtttca aagaattgaa gggaatactg acattttcag aagctacaca
     2281 tgctcctata tccatttatt ttgatttccc tgggaaacct ctggctttga gtattgatga
     2341 tatgttagtg gaagctaact ttattttggc cacattagct gatgaacaaa gtagagcatc
     2401 ttcaccacag tcactgtgtc tttcacagaa acgaaaaagg tcagatctga ttgaaaaaaa
     2461 ggctggcaaa aatgtaactg gccaggccct ggaatgtatt tcaaaaaagc agcaccaaga
     2521 aggctttatc ctaaggagac tctcacaaac atatctgcat tggaaaactg tggcagccct
     2581 gcaatgaaaa gagtggatgg agatgtcagt gaagtatcag aaagcagtgt cagcaacaca
     2641 gaggaagtgc cagggtctct gtgtctcaga aagttttctt gcatgttctt tggagcagtt
     2701 tcttctgacc agcaagaaca cttcaaccac cctttcgaca gtctggcaag agcaagtgac
     2761 agtgaagagg acatgaataa tgtgtgctgc aggaaagaat ttaatggaag tgatgccaaa
     2821 tatttctgta ttatctgagg atccgaattc tgcagatatc cagcacagtg gcggccgctc
     2881 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     2941 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3001 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3061 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3121 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     3181 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3241 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3301 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3361 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3421 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3481 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3541 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3601 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3661 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3721 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3781 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3841 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3901 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3961 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     4021 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4081 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4141 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4201 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4261 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4321 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4381 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4441 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4501 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     4561 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4621 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4681 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4741 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4801 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4861 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4921 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4981 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5041 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5101 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     5161 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5221 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5281 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5341 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5401 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5461 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5521 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5581 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5641 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5701 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5761 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5821 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5881 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5941 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6001 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6061 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6121 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6181 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6241 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6301 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6361 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6421 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6481 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6541 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6601 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6661 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6721 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6781 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6841 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6901 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6961 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7021 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7081 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7141 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7201 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7261 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     7321 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     7381 gatgccgcat agttaagcca gtatctgctc cctgctt
//
