LOCUS       sequence-222602-        3341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express g-AAVS1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3341)
  AUTHORS   Ma S, Lv J, Sun J, Tang P, Li H, Zhou H, Zhang Z, Lin Y, Rong Z
  TITLE     iKA-CRISPR hESCs for inducible and multiplex orthogonal gene 
            knockout and activation.
  JOURNAL   FEBS Lett. 2018 Jun 5. doi: 10.1002/1873-3468.13127.
  PUBMED    29869798
REFERENCE   2  (bases 1 to 3341)
  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: "FEBS Lett. 
            2018 Jun 5. doi: 10.1002/1873-3468.13127."
COMMENT     Sequence Label: pU6-EF-gAAVS1
FEATURES             Location/Qualifiers
     source          1..3341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     672..688
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     673..689
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             706..717
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        725..973
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     725..745
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     896..915
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1156..1176)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1156..1174)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1195..1211)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1195..1211)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1208..1230)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1219..1235
                     /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(1243..1249,1250..1267,1268..1273))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1288..1309
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1426..1443)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1597..2185)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1677..1696)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2356..3147,3148..3216))
                     /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     2979..2998
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3217..3321)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
      661 cgggcccccc ctcgaggtcg acggtatcga taagcttgat atcgaattga aggtcgggca
      721 ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
      781 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
      841 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
      901 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
      961 aggacgaaac accgtcacca atcctgtccc taggtttaag agctatgctg gaaacagcat
     1021 agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     1081 ttttttacgc gttgacattg attattgact agttctagag cggccgccac cgcggtggag
     1141 ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
     1201 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     1261 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     1321 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1381 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     1441 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1501 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1561 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1621 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1681 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1741 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1801 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1861 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1921 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1981 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2041 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2101 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2161 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2221 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2281 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2341 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2401 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2461 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2521 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     2581 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     2641 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2701 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2761 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2821 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2881 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2941 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3001 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3061 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3121 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3181 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3241 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3301 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg c
//
