LOCUS       sequence-376579-        3753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template vector to amplify pieces for creating multiplex 
            sgRNA-arrays..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3753)
  AUTHORS   Cengic I, Canadas IC, Minton NP, Hudson EP
  TITLE     Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated 
            Single-Target Genome Editing in Synechocystis Sp. PCC 6803.
  JOURNAL   ACS Synth Biol. 2022 Aug 15. doi: 10.1021/acssynbio.2c00375.
  PUBMED    35969224
REFERENCE   2  (bases 1 to 3753)
  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. 2022 Aug 15. doi: 10.1021/acssynbio.2c00375."
COMMENT     Sequence Label: 
            pMD19-Cas9_handle-S.pyogenes_terminator-PBBa_J23117-Cmr
FEATURES             Location/Qualifiers
     source          1..3753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    433..454
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     misc_RNA        454..529
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        537..571
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     terminator      589..660
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      676..703
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             811..1470
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(877..896)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     misc_feature    1471..1491
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(1532..1548)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1532..1548)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1545..1567)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1556..1572
                     /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(1580..1586,1587..1604,1605..1610))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1625..1646
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1763..1780)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1934..2522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2014..2033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2693..3484,3485..3553))
                     /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     3316..3335
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3554..3658)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3726..3744
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggt acgcgcggat
      421 cttccagaga ttgaattcgc ggccgcttct agagttttag agctagaaat agcaagttaa
      481 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttttga
      541 cagctagctc agtcctaggg attgtgctag ctaactagag ccaggcatca aataaaacga
      601 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
      661 tactagagtc acactggctc accttcgggt gggcctttct gcgtttatat actagaggta
      721 agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta
      781 tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc
      841 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa
      901 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa
      961 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat
     1021 ccggaatttc gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct
     1081 tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac
     1141 gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac
     1201 ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg
     1261 gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt
     1321 ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag
     1381 gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag
     1441 tactgcgatg agtggcaggg cggggcgtaa tactagtagc ggccgctgca gaatcgtcga
     1501 acggcaggcg tgcaaacttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     1561 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     1621 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     1681 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1741 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1801 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1861 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1921 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1981 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2041 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2101 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2161 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2221 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2281 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2341 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2401 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2461 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2521 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2581 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2641 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     2701 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     2761 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     2821 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     2881 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     2941 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3001 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3061 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3121 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3181 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3241 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3301 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3361 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3421 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3481 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3541 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     3601 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaatgggg gttccgcgca
     3661 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     3721 ataaaaatag gcgtatcacg aggccctttc gtc
//
