LOCUS       sequence-224220-        5787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generates Nonsense mutant clone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5787)
  AUTHORS   Pattnaik BR, Shahi PK, Marino MJ, Liu X, York N, Brar S, Chiang J, 
            Pillers DA, Traboulsi EI
  TITLE     A Novel KCNJ13 Nonsense Mutation and Loss of Kir7.1 Channel Function
            Causes Leber Congenital Amaurosis (LCA16).
  JOURNAL   Hum Mutat. 2015 Jul;36(7):720-7. doi: 10.1002/humu.22807. Epub 2015 
            May 20.
  PUBMED    25921210
REFERENCE   2  (bases 1 to 5787)
  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.22807"; journalName: "Hum Mutat"; date: "2015-07"; 
            volume: "36"; issue: "7"; pages: "720-7"
COMMENT     Sequence Label: c1GFP-W53XKir7.1
FEATURES             Location/Qualifiers
     source          1..5787
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        86..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     544..564
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      632..641
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(638..640,641..643,644..1354)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(638..640,641..643,644..1354)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(683..704)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(944..963)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1291..1312
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2600..2721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2637..2656)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2691..2710
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2728..3183)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2865..2886)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3077..3096
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3210..3314
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3316..3673
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3316..3507
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3339..3359)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3524..3659
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3708..4502
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3708..4502
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3762..3781)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4372..4391
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4690..4709)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4734..4781
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5110..5698
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
      601 tgaaccgtca gatccgctag cgctaccggt cgccaccatg gtgagcaagg gcgaggagct
      661 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
      721 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
      781 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
      841 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
      901 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
      961 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1021 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1081 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1141 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1201 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     1261 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1321 cgggatcact ctcggcatgg acgagctgta caagtccgga ctcagatctc gagctcaagc
     1381 ttcgaattcc gacagcagta attgcaaagt tattgctcct ctcctaagtc aaagataccg
     1441 gaggatggtc accaaggatg gccacagcac acttcaaatg gatggcgctc aaagaggtct
     1501 tgcatatctt cgagatgctt ggggaatcct aatggacatg cgctagcgtt ggatgatgtt
     1561 ggtcttttct gcttcttttg ttgtccactg gcttgtcttt gcagtgctct ggtatgttct
     1621 ggctgagatg aatggtgatc tggaactaga tcatgatgcc ccacctgaaa accacactat
     1681 ctgtgtcaag tatatcacca gtttcacagc tgcattctcc ttctccctgg agacacaact
     1741 cacaattggt tatggtacca tgttccccag tggtgactgt ccaagtgcaa tcgccttact
     1801 tgccatacaa atgctcctag gcctcatgct agaggctttt atcacaggtg cttttgtggc
     1861 gaagattgcc cggccaaaaa atcgagcttt ttcaattcgc tttactgaca tagcagtagt
     1921 agctcacatg gatggcaaac ctaatcttat cttccaagtg gccaacaccc gacctagccc
     1981 tctaaccagt gtccgggtct cagctgtact ctatcaggaa agagaaaatg gcaaactcta
     2041 ccagaccagt gtggatttcc accttgatgg catcagttct gacgaatgtc cattcttcat
     2101 ctttccacta acgtactatc actccattac accatcaagt cctctggcta ctctgctcca
     2161 gcatgaaaat ccttctcact ttgaattagt tgtattcctt tcagcaatgc aggagggcac
     2221 tggagaaata tgccaaagga ggacatccta cctacagtct gaaatcatgt tacatcactg
     2281 ttttgcatct ctgttgaccc gaggttccaa atgtgaatat caaatcaaga tggagaattt
     2341 tgacaagact gtccctgaat ttccaactcc tctggtttct aaaagcccaa acaggactga
     2401 cctggatatc cacatcaatg gacaaagcat tgacaatttt cagatctctg aaacaggact
     2461 gacagaataa ggatccaccg gatctagata actgatcata atcagccata ccacatttgt
     2521 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     2581 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     2641 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     2701 caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     2761 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2821 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     2881 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     2941 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     3001 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     3061 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     3121 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     3181 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3241 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3301 gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     3361 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3421 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3481 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3541 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3601 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3661 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3721 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3781 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3841 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     3901 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3961 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4021 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4081 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4141 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4201 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     4261 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4321 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4381 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4441 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4501 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4561 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4621 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     4681 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     4741 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     4801 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     4861 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     4921 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     4981 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5041 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5101 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5161 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5221 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5281 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5341 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5401 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5461 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5521 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5581 cacgagggag cttccagggg gaaacgcctg ttatctttat agtcctgtcg ggtttcgcca
     5641 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5701 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5761 ctttcctgcg ttatcccctg attctgt
//
