LOCUS       sequence-194886-        5810 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Basic cloning vector for the expression of imaging-based 
            optimization sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5810)
  AUTHORS   Shao S, Chang L, Sun Y, Hou Y, Fan X, Sun Y
  TITLE     Multiplexed sgRNA Expression Allows Versatile Single Nonrepetitive 
            DNA Labeling and Endogenous Gene Regulation.
  JOURNAL   ACS Synth Biol. 2017 Sep 7. doi: 10.1021/acssynbio.7b00268.
  PUBMED    28849913
REFERENCE   2  (bases 1 to 5810)
  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: "ACS Synth 
            Biol. 2017 Sep 7. doi: 10.1021/acssynbio.7b00268."
COMMENT     Sequence Label: psgRNA-ccdB
FEATURES             Location/Qualifiers
     source          1..5810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    123..171
                     /note="synthetic polyadenylation signal"
     misc_feature    185..276
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     225..244
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        305..553
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     305..325
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     476..495
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     577..596
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        577..595
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    625..749
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1178..1483)
                     /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     complement(1280..1299)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1803..2486)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2401..2420
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2487..2589)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2767..2891)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3056..3177
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3067..3086)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3121..3140
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3425..3442)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3596..4184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3676..3695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4355..5146,5147..5215))
                     /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     4978..4997
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5216..5320)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5347..5802
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5434..5453)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5644..5665
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
       61 cagcccaagc taccatgata agtaagtaat attaaggtac gggaggtact tggagcggcc
      121 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
      181 tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca aaataggctg
      241 tccccagtgc aagtgcaggt gccagaacat ttctctatcg ataggtacca aggtcgggca
      301 ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
      361 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
      421 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
      481 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
      541 aggacgaaac accgagagac gctggcttat cgaaattaat acgactcact atagggagac
      601 ccaagctggc tagttaagct atcaacaagt ttgtacaaaa aagctgaacg agaaacgtaa
      661 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
      721 tgtaaaacac aacatatcca gtcactatga atcaactact tagatggtat tagtgacctg
      781 tagtcgaccg acagccttcc aaatgttctt cgggtgatgc tgccaactta gtcgaccgac
      841 agccttccaa atgttcttct caaacggaat cgtcgtatcc agcctactcg ctattgtcct
      901 caatgccgta ttaaatcata aaaagaaata agaaaaagag gtgcgagcct cttttttgtg
      961 tgacaaaata aaaacatcta cctattcata tacgctagtg tcatagtcct gaaaatcatc
     1021 tgcatcaaga acaatttcac aactcttata cttttctctt acaagtcgtt cggcttcatc
     1081 tggattttca gcctctatac ttactaaacg tgataaagtt tctgtaattt ctactgtatc
     1141 gacctgcaga ctggctgtgt ataagggagc ctgacattta tattccccag aacatcaggt
     1201 taatggcgtt tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa
     1261 cggagaccgg cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat
     1321 gcaccaccgg gtaaagttca cgggagactt tatctgacag cagacgtgca ctggccaggg
     1381 ggatcaccat ccgtcgcccg ggcgtgtcaa taatatcact ctgtacatcc acaaacagac
     1441 gataacggct ctctctttta taggtgtaaa ccttaaactg catttcacca gcccctgttc
     1501 tcgtcagcaa aagagccgtt catttcaata aaccgggcga cctcagccat cccttcctga
     1561 ttttccgctt tccagcgttc ggcacgcaga cgacgggctt cattctgcat ggttgtgctt
     1621 accagaccgg agatattgac atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa
     1681 ctgtcactgt aatacgctgc ttcatagcat acctcttttt gacatacttc gggtatacat
     1741 atcagtatat attcttatac cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc
     1801 ttttagtaag ccggatccac gcggcgttta cgccccccct gccactcatc gcagtactgt
     1861 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
     1921 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg
     1981 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
     2041 ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
     2101 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     2161 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     2221 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa ttccggatga
     2281 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc
     2341 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     2401 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     2461 gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac
     2521 tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg
     2581 tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg gcttcccggt atcaacaggg
     2641 acaccaggat ttatttattc tgcgaagtga tcttccgtca caggtattta ttcggcgcaa
     2701 agtgcgtcgg gtgatgctgc caacttagtc gactacaggt cactaatacc atctaagtag
     2761 ttgattcata gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc
     2821 aaaatctaat ttaatatatt gatatttata tcattttacg tttctcgttc agctttcttg
     2881 tacaaagtgg ttgatctaga gggcccgcgg ttcgaacgtc tctgtttaag agctatgctg
     2941 gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     3001 agtcggtgct tttttttcta gagtcggggc ggccggccgc ttcgagcaga catgataaga
     3061 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     3121 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     3181 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     3241 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtcgac cgatgccctt
     3301 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     3361 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctcttccg
     3421 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3481 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3541 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3601 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3661 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3721 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3781 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3841 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3901 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3961 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4021 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4081 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4141 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4201 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4261 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4321 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4381 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4441 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4501 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4561 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4621 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4681 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4741 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4801 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4861 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4921 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4981 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5041 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5101 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5161 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5221 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5281 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5341 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5401 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     5461 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5521 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     5581 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5641 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     5701 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5761 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttgccattcg
//
