LOCUS       sequence-249152-        7419 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 7419)
  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 7419)
  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-FS2
FEATURES             Location/Qualifiers
     source          1..7419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        66..445
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        446..649
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     600..620
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     694..713
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        694..712
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             754..1467
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(754..756,757..1467)
                     /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(796..817)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1057..1076)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1404..1425
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2841..2858)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2847..3071
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3117..3545
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3204..3223)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3414..3435
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3554..3574)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3559..3888
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3739..3874
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3801..3820
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3955..4749
                     /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(4009..4028)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4619..4638
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4923..5044
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4960..4979)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5014..5033
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5093..5109)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5093..5109)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5106..5128)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5117..5133
                     /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(5141..5147,5148..5165,5166..5171))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5186..5207
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5324..5341)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5495..6083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5575..5594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6254..7045,7046..7114))
                     /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     6877..6896
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7115..7219)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7294..7313)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
       61 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
      121 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      181 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      241 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      301 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      361 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      421 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      481 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      541 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      601 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac
      661 tagagaaccc actgcttact ggcttatcga aattaatacg actcactata gggagaccca
      721 agctggctag cgtttaaact taagcttgcc accatgagca agggcgagga gctgttcacc
      781 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      841 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      901 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
      961 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1021 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1081 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1141 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1201 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1261 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1321 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1381 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1441 actcacggca tggacgagct gtacaagggt accatggcag ccatgctgaa gtgcgtgatg
     1501 agcggcagtc aggtgaaagt atttgggaaa gcagttcaag ctctatcacg aattagtgac
     1561 gagttctggc tagacccatc taaaaaaggt cttgctctaa gatgtgtgaa ttcttctcgg
     1621 tcagcatatg gatgtgtcct gttctctcct gtgttttttc agcattatca atggtcagct
     1681 ttagtgaaaa tgagtgaaaa tgaacttgac acaacactgc atttaaaatg caaattggga
     1741 atgaagtcaa ttttgcccat ctttagatgt ctgaattccc ttgaaagaaa tatagagaag
     1801 tgcagaatat tcaccagatc tgataaatgc aaagtagtta ttcaattctt ctacagacat
     1861 ggtattaaaa gaactcataa tatatgtttt caagaaagtc agcctttgca agttattttt
     1921 gacaagaatg tttgtactaa tacgctaatg attcaaccaa gattgcttgc tgatgccatt
     1981 gttcttttta catcaagtca agaggaagtt actcttgctg ttactccact gaatttttgc
     2041 ctcaagagtt ctaatgagga atcaatggat ttgagcaatg ctgtacacag tgagatgttt
     2101 gttggctcag atgagtttga cttctttcaa attggaatgg acactgagat aacattttgt
     2161 ttcaaagaat tgaagggaat actgacattt tcagaagcta cacatgctcc tatatccatt
     2221 tattttgatt tccctgggaa acctctggct ttgagtattg atgatatgtt agtggaagct
     2281 aactttattt tggccacatt agctgatgaa caaagtagag catcttcacc acagtcactg
     2341 tgtctttcac agaaacgaaa aaggtcagat ctgattgaaa aaaaggctgg caaaaatgta
     2401 actggccagg ccctggaatg tatttcaaaa aaagcagcac caagaaggct ttatcctaag
     2461 gagactctca caaacatatc tgcattggaa aactgtggca gccctgcaat gaaaagagtg
     2521 gatggagatg tcagtgaagt atcagaaagc agtgtcagca acacagagga agtgccaggg
     2581 tctctgtgtc tcagaaagtt ttcttgcatg ttctttggag cagtttcttc tgaccagcaa
     2641 gaacacttca accacccttt cgacagtctg gccaagagca agtgacagtg aagaggacat
     2701 gaataatgtg tgctgcagga aagaatttaa tggaagtgat gccaaatatt tctgtattat
     2761 ctgaggatcc gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc
     2821 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     2881 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2941 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3001 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3061 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3121 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3181 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3241 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3301 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3361 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3421 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3481 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3541 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3601 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3661 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3721 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3781 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     3841 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     3901 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     3961 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4021 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4081 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4141 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4201 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4261 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4321 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4381 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4441 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4501 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4561 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4621 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4681 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4741 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4801 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4861 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4921 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4981 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5041 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5101 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5161 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5221 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5281 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5341 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5401 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5461 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5521 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5581 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5641 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5701 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5761 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5821 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5881 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5941 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6001 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6061 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6121 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6181 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6241 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6301 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6361 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6421 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6481 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6541 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6601 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6661 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6721 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6781 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6841 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6901 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6961 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7021 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7081 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7141 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7201 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     7261 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     7321 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     7381 ttaagctaca acaaggcaag gcttgaccga caattgcat
//
