LOCUS       sequence-224555-        7109 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To construct multiple sgRNA expression vector for CRISPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7109)
  TITLE     multiple sgRNA vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7109)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pGL3-U6-XsgRNA-ccdB-EF1a-Puromycin
FEATURES             Location/Qualifiers
     source          1..7109
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        join(349..354,355..372,373..379)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             433..1092
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(499..518)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1434..1739
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1618..1637
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1788..1863
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1961..2078
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2124..3306
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2355..3297
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3254..3274
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3326..3925
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3326..3345)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3822..3842
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4072..4193
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4083..4102)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4137..4156
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4441..4458)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4612..5200)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4692..4711)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5371..6162,6163..6231))
                     /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     5994..6013
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6232..6336)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6363..6818
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6450..6469)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6660..6681
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    6949..6997
                     /note="synthetic polyadenylation signal"
     misc_feature    7011..7102
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     7051..7070
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc gggagacgcc gcattaggca ccccaggctt tacactttat
      361 gcttccggct cgtataatgt gtggattttg agttaggatc cgtcgagatt ttcaggagct
      421 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
      481 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
      541 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
      601 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt ccgtatggca
      661 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
      721 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
      781 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
      841 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
      901 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat
      961 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt
     1021 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
     1081 ggcggggcgt aaagatctgg atccggctta ctaaaagcca gataacagta tgcgtatttg
     1141 cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag tatgtcaaaa
     1201 agaggtatgc tatgaagcag cgtattacag tgacagttga cagcgacagc tatcagttgc
     1261 tcaaggcata tatgatgtca atatctccgg tctggtaagc acaaccatgc agaatgaagc
     1321 ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc
     1381 ccggtttatt gaaatgaacg gctcttttgc tgacgagaac aggggctggt gaaatgcagt
     1441 ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg
     1501 atattattga cacgcccggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt
     1561 cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca
     1621 tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc
     1681 tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa
     1741 tgtcaggctc ccttatacac agccagtctg caggtcgacg cgtctccgtt ttagagctag
     1801 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     1861 tgcttttttt aaagggcccg tcgactgcag aggcctgcat gcaagcttgg cgtaatcata
     1921 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     1981 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     2041 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     2101 gagatccact ttggccgccg tgaggctccg gtgcccgtca gtgggcagag cgcacatcgc
     2161 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt
     2221 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     2281 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     2341 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt
     2401 atggcccttg cgtgccttga attacttcca cgcccctggc tgcagtacgt gattcttgat
     2461 cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct
     2521 tcgcctcgtg cttgagttga ggcctggctt gggcgctggg gccgccgcgt gcgaatctgg
     2581 tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga
     2641 tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg
     2701 cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc
     2761 acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct
     2821 caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg
     2881 gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc
     2941 cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca
     3001 cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag
     3061 taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta
     3121 ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa
     3181 gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga
     3241 tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg
     3301 tcgtgaggat ccaccggagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac
     3361 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     3421 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     3481 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     3541 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     3601 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     3661 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtgtcgcc
     3721 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     3781 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     3841 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     3901 catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag
     3961 gagcgcacga ccccatgcat cggtaccttt aagaccaatg acttacaagg cagctgtaga
     4021 tcttagccac tttctagagt cggggcggcc ggccgcttcg agcagacatg ataagataca
     4081 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     4141 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     4201 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     4261 agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc gtcgaccgat gcccttgaga
     4321 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     4381 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc
     4441 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4501 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4561 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4621 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4681 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4741 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4801 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4861 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4921 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4981 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5041 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5101 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5161 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5221 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5281 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5341 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5401 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5461 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5521 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5581 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5641 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5701 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5761 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgattgttgt
     5821 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5881 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5941 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6001 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6061 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6121 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6181 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6241 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6301 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6361 tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     6421 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     6481 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     6541 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     6601 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     6661 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     6721 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     6781 taacgcgaat tttaacaaaa tattaacgct tacaatttgc cattcgccat tcaggctgcg
     6841 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc ccaagctacc
     6901 atgataagta agtaatatta aggtacggga ggtacttgga gcggccgcaa taaaatatct
     6961 ttattttcat tacatctgtg tgttggtttt ttgtgtgaat cgatagtact aacatacgct
     7021 ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagt
     7081 gcaggtgcca gaacatttct ctatcgata
//
