LOCUS       sequence-481699-        6025 bp    DNA     circular SYN 01-AUG-2025
DEFINITION  HITI insert construct with Ckm gRNA and hF9 transgene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6025)
  AUTHORS   Padmaswari MH, Bulliard GN, Agrawal S, Jia MS, Nelson CE
  TITLE     Precision and efficacy of RNA-guided DNA integration in 
            high-expressing muscle loci.
  JOURNAL   bioRxiv [Preprint]. 2024 Mar 19:2024.03.18.582796. doi: 
            10.1101/2024.03.18.582796.
  PUBMED    38562818
REFERENCE   2  (bases 1 to 6025)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Mar 19:2024.03.18.582796. doi: 
            10.1101/2024.03.18.582796."
COMMENT     Sequence Label: p-HITI-F9-Ckm
FEATURES             Location/Qualifiers
     source          1..6025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    835..856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(871..876,877..894,895..901)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    909..925
                     /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)."
     primer_bind     914..936
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     933..949
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     933..949
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     968..988
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        970..988
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    3940..4074
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3964..3983)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4018..4037
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4180..4199)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4181..4199)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4209..4226)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4209..4225)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4218..4240)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4367..4822
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4454..4473)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4664..4685
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4848..4952
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4953..5021,5022..5813)
                     /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     complement(5171..5190)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5984..6025,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      781 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      841 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      901 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      961 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctcccct atgtgatcct
     1021 gctggggact atgtccccta ggttcctggg gagggaggac gtcggggcca gccaggctta
     1081 ccccccctgc cgctgagact tctgcgctgg tgcaccgcgc ctcttcgcgg tctccctgtc
     1141 cttgcagaga accggtcagg ccaccatgca gcgcgtgaac atgatcatgg cagaatcacc
     1201 aggcctcatc accatctgcc ttttaggata tctactcagt gctgaatgta cagtttttct
     1261 tgatcatgaa aacgccaaca aaattctgaa tcggccaaag aggtataatt caggtaaatt
     1321 ggaagagttt gttcaaggga accttgagag agaatgtatg gaagaaaagt gtagttttga
     1381 agaagcacga gaagtttttg aaaacactga aagaacaact gaattttgga agcagtatgt
     1441 tgatggagat cagtgtgagt ccaatccatg tttaaatggc ggcagttgca aggatgacat
     1501 taattcctat gaatgttggt gtccctttgg atttgaagga aagaactgtg aattagatgt
     1561 aacatgtaac attaagaatg gcagatgcga gcagttttgt aaaaatagtg ctgataacaa
     1621 ggtggtttgc tcctgtactg agggatatcg acttgcagaa aaccagaagt cctgtgaacc
     1681 agcagtgcca tttccatgtg gaagagtttc tgtttcacaa acttctaagc tcacccgtgc
     1741 tgagactgtt tttcctgatg tggactatgt aaattctact gaagctgaaa ccattttgga
     1801 taacatcact caaagcaccc agtcatttaa tgacttcact cgggttgttg gtggagaaga
     1861 tgccaaacca ggtcaattcc cttggcaggt tgttttgaat ggtaaagttg atgcattctg
     1921 tggaggctct atcgttaatg aaaaatggat tgtaactgct gcccactgtg ttgaaactgg
     1981 tgttaaaatt acagttgtcg caggtgaaca taatattgag gagacagaac atacagagca
     2041 aaagcgaaat gtgattcgaa ttattcctca ccacaactac aatgcagcta ttaataagta
     2101 caaccatgac attgcccttc tggaactgga cgaaccctta gtgctaaaca gctacgttac
     2161 acctatttgc attgctgaca aggaatacac gaacatcttc ctcaaatttg gatctggcta
     2221 tgtaagtggc tggggaagag tcttccacaa agggagatca gctttagttc ttcagtacct
     2281 tagagttcca cttgttgacc gagccacatg tcttcgatct acaaagttca ccatctataa
     2341 caacatgttc tgtgctggct tccatgaagg aggtagagat tcatgtcaag gagatagtgg
     2401 gggaccccat gttactgaag tggaagggac cagtttctta actggaatta ttagctgggg
     2461 tgaagagtgt gcaatgaaag gcaaatatgg aatatatacc aaggtatccc ggtatgtcaa
     2521 ctggattaag gaaaaaacaa agctcactta atgaaagatg gatttccaag gttaattcat
     2581 tggaattgaa aattaacagg gcctctcact aactaatcac tttcccatct tttgttagat
     2641 ttgaatatat acattctatg atcattgctt tttctcttta caggggagaa tttcatattt
     2701 tacctgagca aattgattag aaaatggaac cactagagga atataatgtg ttaggaaatt
     2761 acagtcattt ctaagggccc agcccttgac aaaattgtga agttaaattc tccactctgt
     2821 ccatcagata ctatggttct ccactatggc aactaactca ctcaattttc cctccttagc
     2881 agcattccat cttcccgatc ttctttgctt ctccaaccaa aacatcaatg tttattagtt
     2941 ctgtatacag tacaggatct ttggtctact ctatcacaag gccagtatca cactcatgaa
     3001 gaaagaacac aggagtagct gagaggctaa aactcatcaa aaacactact ccttttcctc
     3061 taccctattc ctcaatcttt taccttttcc aaatcccaat ccccaaatca gtttttctct
     3121 ttcttactcc ctctctccct tttaccctcc atggtcgtta aaggagagat ggggagcatc
     3181 attctgttat acttctgtac acagttatac atgtctatca aacccagact tgcttccgta
     3241 gtggagactt gcttttcaga acatagggat gaagtaaggt gcctgaaaag tttgggggaa
     3301 aagtttcttt cagagagtta agttatttta tatatataat atatatataa aatatataat
     3361 atacaatata aatatatagt gtgtgtgtat gcgtgtgtgt agacacacac gcatacacac
     3421 atataatgga agcaataagc cattctaaga gcttgtatgg ttatggaggt ctgactaggc
     3481 atgatttcac gaaggcaaga ttggcatatc attgtaacta aaaaagctga cattgaccca
     3541 gacatattgt actctttcta aaaataataa taataatgct aacagaaaga agagaaccgt
     3601 tcgtttgcaa tctacagcta gtagagactt tgaggaagaa ttcaacagtg tgtcttcagc
     3661 agtgttcaga gccaagcaag aagttgaagt tgcctagacc agaggacata agtatcatgt
     3721 ctcctttaac tagcataccc cgaagtggag aagggtgcag caggctcaaa ggcataagtc
     3781 attccaatca gccaactaag ttgtcctttt ctggtttcgt gttcaccatg gaacattttg
     3841 attatagtta atccttctat cttgaatctt ctagagagtt gctgaccaac tgacgtatgt
     3901 ttccctttgt gaattaataa actggtgttc tggttcatgg atccagacat gataagatac
     3961 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     4021 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     4081 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttagtcg
     4141 acccctatgt gatcctgctg gggactggta cccaattcgc cctatagtga gtcgtattac
     4201 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4261 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4321 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     4381 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4441 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4501 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4561 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     4621 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4681 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     4741 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4801 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4861 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4921 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4981 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5041 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5101 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5161 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5221 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5281 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5341 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5401 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5461 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5521 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5581 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5641 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5701 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     5761 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5821 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5881 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5941 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6001 tgcgcgtaat ctgctgcttg caaac
//
